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If sort of this stuff between us and, you know, as they said, we think we met in the mid to late seventies at a meeting in Cologne,

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but in fact, with a missing us of time and the number of pints we've drunk together over that period.

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And of course, we're getting older. Our brains are not terribly functional anymore.

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Our memories are not very functional anymore. So.

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David got his first lectureship in the University of Sussex, which was the School of Biological Sciences,

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which was set up by John Maynard Smith, who is probably the most incisive brain I've ever come across.

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And who was who worked with JBS Holden and who introduced James theory into into biology.

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And the second person on the right is Neville Simons, who was a tea fall person, a converted physicist and who worked with that with Schrodinger.

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And so there we are, a young chicken in his prime from seventy one to nineteen eighty, and he set up a plasma lab in Sussex.

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And in fact, at the time, plasmids were getting a pretty bad press.

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This is from Mike Smolensk. It's the final chapter of a Cold Spring Harbour meeting in 78 and where he was under this table.

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So which is the fashionable replication of the what is the professional mentality of the of the personalities?

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And so we have got four physicists tough minded lambda physician, tender minded cosmonauts, entrepreneur.

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This is the first cloning, mind boggling me DNA, which is the human genome, and it brought in mining but poor plasmids, epidemiologists mindless.

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But I think we we all know that plasmids, which was Dave's first love, have proved to me very important really.

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And they're important in acquisition and dissemination of genetic material.

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The key to this discovery of transpose elements transposition,

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this is to validate these studies and plasmid based transposons play a major role in plasmid behaviour and the impact on genomes.

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And there's a more modular organisation which makes the frontiers between these plasmids and transposons,

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both of which they've worked on rather fuzzy.

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And lastly, the plasmids are relatively good and model system to start analysing chromosomes.

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So what is going through some of his career, and I think he's had this secret separate life?

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I mean, you know, I came across this in the Daily Mirror.

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Not that I read the Daily Mirror very often. And then I came across this.

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I mean, yeah, he swears it's fake news. Yeah.

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And I tend to believe him, in fact. But but I think.

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What I remember about Dave, is this sort of picture where we had lots of discussions in pubs over a beer.

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And that is so I tend to believe what he said about his the fake news.

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And well before I finish here, I expect all of you have followed the usual following approach to science and you get data.

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Which comes a turns into information which generates knowledge.

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Sometimes you get insights. Which become wisdom.

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And if you don't follow this, what happens is you get conspiracy theories.

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And sorry, that that's a quote from the tiger lilies. Anyway, so let's get on to we've got a packed programme.

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There are three young guns or not so young guns now from the early days.

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So Claudia Stone, who was an undergraduate? 1972.

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I think that's right. You and Gordon Dougal, Ph.D., and nineteen seventy seven.

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And Lorraine Symington. I couldn't find the date she did her undergraduate at Sussex and then started her Ph.D. in Sussex and ended up in Glasgow.

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So she sort of straddles not struggles this session and the next.

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And I should just congratulate Lorraine because she's just been elected to the National Academy.

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And with that, Claudia. Thanks very much.

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It's an enormous pleasure to be here, and actually, after all that wait,

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we didn't know when it was going to happen, but to be here in person is a particular pleasure.

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And actually, to see Dave and everybody else who has connexions with him is just incredible.

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So it made me think of many things in the past that have got water under the bridge in different ways.

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See if I can get this to work. Hopefully it will react.

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Well, not. Then we go.

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So the title of Dave's symposium on mental lectures and so on a molecular detective make me think that in a sense, we're all detectives.

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I think we're all asking questions about who did, who done it and and how does it work.

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But in a sense, it brings us all together in many different ways and again, thinking back to Sussex.

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Thanks very much for all the introductions already, but. Oh.

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What happened? It brings us back to those days where some of those pictures we've seen already

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here is one of one of the things coming in as an undergraduate in 1972.

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I was very green in those days.

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I really didn't know what to expect at all doing biological sciences and and and this was a lecture theatre touch, nothing.

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So lecture theatre, where we had many lectures, including from Dave at the time,

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one of the interesting issues here was that the lower floors of the building,

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this part of the building were occupied largely by the down to earth plasmid molecular biologist in the early days of molecular biology.

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And then as you moved up the building into the upper floors, we reached the geneticist like Jemmy Sang,

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who was alternating with with John Smith as head of school,

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and Robert Whittle was my personal tutor doing Drosophila genetics in the middle here in this

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middle floor and then on the top floor where the more up in the clouds John Maynard Smith,

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Brian Goodwin, who I ended up doing my Ph.D. with eventually.

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And in a sense, coming in as an undergraduate, we're confronted with this three dimensional map of of of complexity.

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And I'm from the Earth to the clouds and and the choice of where do you want to be?

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Do you want it to be rooted in the on on the ground and and dealing with reality

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or more with the imaginary world of of things that we didn't really understand,

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like evolution and you couldn't do experiments.

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And so but it all came together in a way, and it all came together here, and it was really quite interesting to be confronted with all this.

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One of the things that I really remember from those days that being sort of there,

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I found the the the cellular tissues and embryos and organisms much more natural in terms of understanding

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than the very early days of molecular biology when we had to purify it goes one in the lab.

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In fact, it was Dave who ran those practicals. It was very memorable.

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But Neville Simons and Alan Raymond and several other people, Sydney and so on,

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gave us lectures at the time couldn't understand a word that was saying because it was all very,

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very excited by their own research, but they didn't make it accessible. When Dave came and gave the lectures,

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it suddenly all clicked and they realised that actually molecular genetics was

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something that you really did want to know to apply to whatever you wanted to get into.

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And then at the same time, it was all going to genes and and how genes interact with each other,

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the level of mega years and so on from John and Brian.

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And then again, this three dimensional lots of things. So I I couldn't resist being fascinated by looking at the biology, though at the same time.

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And one of the things that really captivated me at the time is just embryos, just looking at embryos and what happens.

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The idea that a system that has thousands or even more than thousands of cells where every cell has the same genetic information,

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and yet they do something like that in a very short time where you go from a desk of something to suddenly all the cells know what to do,

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and in a period of, say, 24 hours or something like that, you suddenly have organisation.

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So the question arises, is where is that complexity?

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How where is the programme that drives this if all the cells have exactly the same genetic information?

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In fact, it starts.

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So this is actually a chick embryo, which is what I ended up working on on the upper floors with Brian and Grant Goodwin and for my Ph.D. later on.

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And it starts out as a disk like this with no symmetry.

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But if you film it, you find that at one end of the embryo, the cell suddenly go down there and they generate the structure,

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which is called the primitive street from where the embryo will arise later.

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So the question that fascinated me right from the beginning from those undergraduate days is how how do these cells know what to do and how?

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How do they communicate and how does that structure get generated?

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Now, one of the things that's really extraordinary in higher vertebrate embryos, it doesn't happen in flies or frogs or anything like this.

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It's just human embryos to do this and birds do, too.

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If you take an embryo at that stage at the beginning of that second movie and you cut it into pieces,

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the embryo has about 50000 cells and about time, every piece will generate a complete embryo.

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So that tells you already that there's some communication between those cells that have

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the same genetic information whereby a cell here knows that it has to do something,

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whereas a cell in this location that has the ability to do the same doesn't do so if there's another one present several millimetres apart.

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So what is that information and how does that relate to then the behaviour of the genes that

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is going to determine how those cells will behave at the level of what happens inside?

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And that's the connexion between the.

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It was a Sussex in that way, and so this is what really, really captivated me so well, it was a Colombian effort.

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We we collaborated with Jane Doe and Tom just about time,

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and we actually cloned a gene that turned out to be the first indicator of polarity very early on.

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It's a member of the beta family called GDF one or Page one.

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And it turns out that if you miss express this gene anywhere in the embryo, you can generate another embryo somewhere else without cutting.

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And so that was really the beginning of of trying to understand what's happened.

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But at the same time, if you look at an in situ hybridisation of that where the gene is expressed and

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you think of what happens to the rest of the embryo in the cutting experiment,

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that makes it even more dramatic in that these cells do not express that gene.

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And yet when you remove one part, somehow they start to express it and begin the whole process again.

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So over about 20 years or so of subsequent work since isolating that gene, we came up with a number of signals I'm not going to bore you with.

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This is just a series of interactions between cells that are secreted.

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Molecules are flow here and everywhere that sort of instruct what's what things happen.

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But that doesn't give you any understanding about the communication or about the gene regulation at all.

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So what I've been doing since then in the last 10 or 15 years is really to try

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and go back to the lower floors at Sussex again and to try and understand what's

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going on at the level of the inside of the cell and how the gene regulation

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takes place in response to this more classical embryology kind of approach.

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So where is the computer programme? How does it work?

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So if you cut an embryo in half and just isolate the upper half with it, normally the embryo doesn't, doesn't begin to form randomly.

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You get expression of one or Judith one, either on on the left.

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Either on the left or on the right hand corner of this piece, I know whether this is not behaving very well.

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We'll be able to do it on the screen, either on the left or on the right. And it's run of 50 50.

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Martha Stewart, well, we have to give this to the other. So we did a screen and embryo by embryo and we did a differential screen using

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RNA sequencing to see which genes are co-expression with BACE1 in embryos,

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where it's on the left or on the right, regardless of the side,

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and then isolate genes that are expressed the same way or the opposite way so that we could find inhibitors and repressors and induces.

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And we came up with two classes of genes some that were like this one that were like potential induces.

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And some that had the exact opposite expressed everywhere, except where the which one is expressed.

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And this was a little clue that that suddenly started to break things up.

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And so we thought so. These two are transcription factors that turn out to be quite important.

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They really do act as repressors.

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If you expressed if you miss Express Cell one and it three repressors in this domain, you block the formation of the axis.

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And if you block that expression somewhere else, you initiate axis formation.

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So it works really quickly. But this work at the level of the inside of the cell.

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How do these things get coordinated?

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So how does it know what it's going to if the half of the embryo is going to express, start doing the embryo forming on the left or on the right?

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How do the cells know that they coordinate the disappearance of the repressors and the appearance of the induces in the same place?

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And this is where we go right into the DNA again.

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The first insight that we've had into what the computer programme might look like.

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And it turns out that the gene that encodes one or glia form in the genome is on chromosome 19.

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In the human and adjacent gene is called UPF one, which encodes a helicase, an RNA helicase which degrades RNA for basically for licencing.

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So it it it checks for errors and transcription, and any errors are cleaned up.

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But there are also some targets RNA targets.

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So if you perform, there are not errors, but are actually arenas that are specifically targeted but upfront don't know very much about it yet.

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These two genes like to be apart. The reading frames is really very, very, very close.

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And before we realised the importance of UPF one,

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we were trying to find by various ways enhancers that control the expression of which one didn't want to find out how the regulation worked.

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And we came up with six enhancers of which two of them we call it really five with red arrows are to turn out to be Oops.

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It's a life of its own turn out to be. Important in, but.

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OK. Right. Can replicate the expression of the GDR, one veg, one in the normal embryo.

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But then given that upfront is an RNA helix that will degrade RNA,

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we wondered whether maybe these two are controlled by the same enhancers that share the unconscious to the same promoters.

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And therefore,

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that would be a mechanism by which the the the domain of expression of the upper one and the domain of expression of which one might be the same.

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So indeed, and in situ as you find it up if one is expressed the same as which one, and that is also the domain where these other ones are absent.

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So could it be that UPF one is in fact another making a case that degrades these arnaiz

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ensuring by code regulation that the same cells lose their precious and gain the inducers?

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And. So this is very much in progress. None of this is published and we're making good progress.

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I'll show you a couple of things very, very quickly of the kind of things that we've been doing.

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This is going back to again, the lower floors of Sussex.

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We tried to see whether indeed it's true that the same enhancers are recruited to the promoters of both genes to regulate their expression.

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So we did come to see micro-cap to see method.

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And this was this is a method designed by you and young in my lab, a postdoc who is very talented and coming up with amazing techniques.

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And it turns out that one of these enhancers E three that replicates the expression of each one in the embryo recruits both promoters of that UPF one,

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which reads this way and the other strand GDF three misunderestimated should be if one goes the other way in the genome.

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The to the two promoters are recruited to the same enhancements based upon this, and we have some data now with ANA immunoprecipitation.

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But if one does indeed bind to those other names we're now doing, those experiments are very much in progress.

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Very quickly, I'll touch a couple of other things to bring this back up again to the embryo and the genes.

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Another postdoc in the lab called him Chile, who's from Korea originally.

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He did some nice and biological experiments and bore you with but,

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suggesting that the information for which side is doing something already in the embryo flows through the outside of the embryo,

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not through the middle. And that correlates with the expression of different gut junction proteins that connect cells to each other.

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In the middle of the embryo, you have one at one set of gut junction proteins that connexion forty three and round the outside connexion 26 and 30.

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And these two types of cells cannot communicate with each other through things that flow between cells.

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So these cells will communicate with each other, but not this way.

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So we wondered whether something like a small ion or small molecules of some sort might flow through the cells and b,

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the informative thing, the informative signals. One possibility is calcium.

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Just recently, we'll be doing some calcium imaging. And to our surprise, this is almost like action potential.

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So this is just a single cell sheet epithelial cells.

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And if you imaged calcium, you see something like this. The frame interval in these movies is one second.

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So this is really like action potentials in epithelium cells. And there's a lattice of cells for the costumes just flowing in between.

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That seems to regulate the expression of it three and so one via a BMP pathway where the outside of the embryo communicates to the inside.

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So that's again, another little piece that brings us back to the sort of middle to the upper floors of the building.

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And finally, going to humans, is this relevant to human twinning a tool?

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And there's two little snippets that have come up again and oops, this is more of the genes and the genetics in the molecular genetics.

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I originally come from here from Uruguay. I was born in Montevideo,

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which is just bottom of the map just before going to Sussex and not very far from the border between southern Brazil and Argentina.

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Very close to Sioux Falls, which is a spectacular place,

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is a tiny community of German immigrants that went to Brazil in the 1830s so more than 200 years ago.

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Almost 200 years ago and settled there,

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and there's a community of about a thousand fifteen hundred people where the proportion of twins is actually astronomical,

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so this little tiny town called Candida Godoy actually a sub part of the town called Venus home Pedro,

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where these this is a reunion that happens every two years.

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And there's all of these are twins. This is a tiny little church for this little village, which is basically one st.

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And on the day we were there in 2016 to sample these people,

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there was a a service on a Saturday morning and there was a pair of twin priests that baptised pairs of twins that had been born like this?

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This born in the previous two years, simultaneously, it was like Monty Python.

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It was just extraordinary. And there are some triplets and many identical twins in the town.

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The pedigrees look phenomenal. This these are some pedigrees, and you can see twins, students like us running in families.

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In fact, it turns out from a visit that is just really for families that have twins and they're all connected with each other.

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So we decided to do some of the genetics. This is just starting to come out in our study.

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But this and another population in India that we've been studying particularly

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well are now giving us a couple of really good regions where there are some,

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there are some very good, very good linkage in association. So in this case, in Brazil, we actually are now this.

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Since this slide was made, we're now down to one region on chromosome 12 for for Brazil that it's very high.

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Again, this is slightly old slide.

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But in this region, the the the apparent mutations that seem to be the region which is causative is there are no there's no coding sequences at all,

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but they're likely to be tied to chromatin structures that associate several genes together.

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So we're trying to see which genes are regulated by this. One of the genes is went one and then maybe an interesting and interesting candidates.

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Meanwhile, I've been collaborating. This is my final slide.

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Meanwhile, I've been collaborating with a worldwide consortium that's looking at twin databases throughout the world and also doing epigenomics.

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And in this one, again, going back to the lower floors of the epigenomics, this this was one main coordinated by what was mine in the Netherlands.

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And this is impressive is about to come out, probably next week or the week after Nature Communications.

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And what we've uncovered by doing this consortium study of putting all the data,

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all the data together from many different populations, we found something extraordinary,

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which is that if you were a an identical twin, if you had an identical twin brethren,

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brother or sister, your DNA is modified for life at that point during doing and development.

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It's an extraordinary thing, so let me see your blood cells or your brain cells or your fibroblasts or contain a mark

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that you retained for the rest of your life to say you were once an identical twin,

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even if you don't know it, you're your brother or sister may have died in neutral and you still retain this epigenetic mark.

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So there are 83 such marks and the monozygotic twins that are really, really similar.

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So they shared an I.M. on an accordion. Some one as I got no amniotic twins have more of these marks and a stronger

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correlation than the ones that are presumably arose earlier in development.

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So these are probably the ones that are closest to our experimental system.

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So that's where we're at now, and I think it brings back really very much to what the Sussex building was like,

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the excitement of the time of seeing biology and then being exposed from the ends, the clouds of the Earth and Dave's practicals and lectures.

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I have never forgotten and met again at a dinner in Oxford a few years back and I just thought, they are amazing.

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This is really where it all comes from. So thank you very much, Dave.

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It's been an amazing experience. Was the twin locus are the dominant trait, so it was one of the populations.

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It appears to be dominant or dominant with a complex, complex phenotype manifestation of the phenotype.

235
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And so this is not absolutely dominant classically, but it is.

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It is at least another population that we have in India,

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that we've studied quite a much larger population than this one is seems to be recessive and probably complex with more than one locus.

238
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So there's all sorts and it's a maternal gene.

239
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So this one is. The it's very complicated because.

240
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Yeah. I don't think I can answer that in the in the Brazilian population because it's not not big enough, but yeah, it.

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Yes. I mean, it doesn't have to be maternal because they're identical twins,

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is that the genome of a zygote rather than the genome of the mother of so and non non-identical twins.

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Does I go to twins? It affects ovulation and so it has to be the genome of a mother fixed up.

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But there is a complexity because we're now finding that the two types of twins often go together in certain populations,

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suggesting that there is some environmental effect as well of the uterus and the

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likelihood that the foetus will survive might affect the incidence of twins second.

247
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Secondly, on top of that, which complicates everything. Thank.

248
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No moral or selective arming deprivation.

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Well, it's one aspect of that is that we're the only mechanism you still get based on transcription or atomic changes of your look.

250
00:27:29,360 --> 00:27:33,800
Yes, we have and you do get nation transcription of the target genes on both sides.

251
00:27:33,800 --> 00:27:41,940
Yeah. Oh yeah. In fact, the ritual in itself starts out on both sides and then one side wins out.

252
00:27:41,940 --> 00:27:48,670
So it's quite dynamic. Yoyo suggestion that you.

253
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PFOA and GDF one would be co regulated as it were and both activated by the same enhancer.

254
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What did you do, do you know whether that enhancer is required for both of those genes?

255
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You mentioned that though you had capture C suggests interacting with the same answer.

256
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The implication is that it's required. We'd love to know that.

257
00:28:12,610 --> 00:28:19,910
Unfortunately, we don't need any. Isn't.

258
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So just quickly, I was curious whether there were any genes strongly associated with twins incidence and in domesticated animals,

259
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specifically sheep, where twins are considered very desirable and not yet for identical twins.

260
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They're probably the most dramatic animal in that sense is the armadillo,

261
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the nine banded armadillo that has obligate quadruplets of every pregnancy, and it does it by splitting twice so the embryo splits.

262
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I mean, essentially makes two embryos initially and then two other sites of embryo formation at right angles.

263
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And so it ends up with quadruplets from a single blast of them that then develop independently.

264
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And the genome of the dustups armadillo is just starting to come up, come out.

265
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We're looking at correspondence between all the sites that we're getting through

266
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the different genetic studies to see if there's any correspondence that not yet.

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So my name's John Doogan, I was actually date's second joint second day student, and I'm now at the University of Cambridge,

268
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but I was undergraduate at Sussex and I was quite unusual in that I had my first child when I was an undergraduate,

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not our second child when I was a student. So I saw the mix tried to be it to learn, to be a father with being a pay station.

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But my first day I walked into, I actually applied to do a pitch day with Neville Simons,

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and I arrived the first day of my pitch day and Nablus said, Well, actually, I'm going on sabbatical.

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We decided to change our supervisor. And you can imagine that nowadays, but I think I was to just take it in my stride.

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I didn't really think too much of it. And then they said, you're working with a guy called the Sharat.

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I thought, Well, that's interesting. And then he asked, David could remember this, that I was still in the corridor list.

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I've been told I saw this guy, long haired guy, coming down the corridor and walking towards me, and it was Dave.

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And I think we thrown together really. And, you know, that was the luckiest day in my life, actually, you know, some things happened by serendipity.

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It was walked in and and I was so naive, I didn't really know what the day was, and I'm sure it's a terrible space station at first.

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But these are the most special, one of the most special period of my life because all we ever did was we sat around and I

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say that I've just taught science and they would talk about holiday mode you talk about.

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They talk about how do they recombine, how plasmids replicated.

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I thought that was that was what I thought it was say it was all about.

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But in reality, there's very few occasions you'd ever get this incredible person in front of you.

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So knowledgeable, so eloquent about the way he said things. And you know,

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he brought me from this level to this level and doubting and really that the science is the is the core of everything you're actually doing.

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David, I think the thing you've kept going, I never believe that you'd be able to keep your career going just by working on Basic Science,

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because there's always a push to be more and more applied that they've kept the

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purity of his stay all the way through his career is a remarkable achievement,

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and I thank you daily for setting me on the road really to.

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My career, so this is me actually as an undergraduate,

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and I was so typical of the times and so my stay really was all about having a conversation with Dave and suggested X,

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Y and Z, and there was other guy in the lab, Rob Halliwell, who turned out to be went eventually to make millions.

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That's because he was one of the first science to Kyron on the patent for the hepatitis

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B invention of making the hepatitis B vaccine in yeast went off to be multimillionaire.

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I never quite achieved that status. Good luck to him, but I work with it.

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And really, it was the time when we were looking at small pieces of DNA like Cody one,

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but also transposed onto really define the hedges and Jacobs defined transposons what they were meant in molecular entity.

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And they was onto this immediately and had the idea about trying to look at the mechanisms by which transportation worked.

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And I did a lot of work. We had a paper called Doogan and share it, which is highly scientific because they made this strain DD's full term,

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which is a mini cell strain which transformed the ability to say proteins off plasmids.

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We actually saw that the very first proteins from a transpose and we saw resolve phase, which Dave identified,

301
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and we also eventually could see the transposes, although we didn't really we couldn't map the transposes at that time.

302
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But also, we were developing methods for mapping plasmids for the first time when I started the day,

303
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we didn't really know where any genes were on the call, the one plasmid. By the end of it, we had matched a lot of genes, actually.

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And Dave also got this concept of mobilisation not only in origin of replication,

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but the origin of transfer all the plasmid which Andrea Twigg and David define.

306
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You know, these real breakthrough programmes that had a big implication because at this time is when

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recombinant DNA was just been identified and a lot of the derogation designed to disable vectors,

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the concepts of how to disable a WorkSafe that came out of Dave's head and at today's laboratory.

309
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And if people really recognised that,

310
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but may I say by hook or by crook was very successful and I was very lucky and I thought it was all about basic science.

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I thought plasmids were all about basic science and about learning, how to understand recombination and learning how to understand transposition.

312
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And this is the group, actually, this is actually me as a station where stood outside the I know if you're on here,

313
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Claudia, because you were in biology. So the days of the back end that was about a Gary Warren elementary.

314
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Gary was your fourth station. Brilliant.

315
00:34:17,660 --> 00:34:25,140
He actually unfortunately died about 10 years ago, and he was very instrumental in shaping a lot of these people.

316
00:34:25,140 --> 00:34:28,950
You know, I remember very well was Oliver Darlington. At the end.

317
00:34:28,950 --> 00:34:38,760
It was an absolutely fantastic group of people is a real privilege to be in amongst that, that group of people.

318
00:34:38,760 --> 00:34:42,090
This is Kelly, who was the president of the students union over here.

319
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I've got to stay in line with the picture. I read about him in the Sun about 20 years later.

320
00:34:47,670 --> 00:34:52,400
I won't say why, but he was a real character. That's. So let's go on.

321
00:34:52,400 --> 00:35:01,220
So what happened then was I actually myself, we're in competition a bit group with a laboratory run by a guy called Stanley Falco.

322
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And worst time was also becoming interested in transposition, and he was a professor at the University of Washington in Seattle.

323
00:35:09,320 --> 00:35:13,190
They went on to get Alaska Pride. They went on to be the most having the people in the world.

324
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I met him at a meeting and I went to a luncheon. That's my first ever trip abroad at 24, so Toronto.

325
00:35:20,020 --> 00:35:25,430
But it's done. Falco and he had this book out called Multiple Infectious Drug Resistance.

326
00:35:25,430 --> 00:35:30,800
The idea that they put into my head was that drug resistance is actually an infection.

327
00:35:30,800 --> 00:35:35,780
People think about drug resistance associated with different bacteria. It's actually an infectious agent in its own right.

328
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It spreads the moves between organisms and an infection and almost a cancerous way,

329
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and standard written this position paper, which was influencing government policy around the use of antibiotics in animals.

330
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But it was really not in the public mind. At that stage, it was a curiosity, was an entity, but it wasn't really dominant.

331
00:35:57,090 --> 00:36:04,690
So that was the context of my Ph.D. in 1974 to seventy seven.

332
00:36:04,690 --> 00:36:13,120
And I took this image, if you remember this image day when I took this, we used to do day mapping by hetero duplex analysis, not by doing DNA digest.

333
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I'm pretty sure I run the first ever agarose gel of a plasmid DNA in the UK.

334
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I can imagine the uptake said. We got this new technology agarose gel at the phrases I pull the agarose gel ran

335
00:36:25,900 --> 00:36:29,380
it and we could actually see DNA as a band on the gel for the very first time.

336
00:36:29,380 --> 00:36:36,520
Before that, we have to use sucrose gradient or caesium chloride, and I can tell you it takes hours to actually make these images.

337
00:36:36,520 --> 00:36:45,220
Geoff Wilson set this technology up for T4. But well, there's this way you just disassociate two pieces of DNA and you allow that to re hybridised.

338
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One has a mercury transposed on it.

339
00:36:47,410 --> 00:36:54,460
One has a antibiotic resistance T and one I remember the first transpose and the beta LATAM's and you use the stem loop.

340
00:36:54,460 --> 00:37:00,760
And up to this is the double stranded DNA. This the single stranded loop and the inverted repeat.

341
00:37:00,760 --> 00:37:04,480
It's a double strand. So that was how that to you. I think you got the equaliser.

342
00:37:04,480 --> 00:37:09,430
Well, we're looking Carly one had one equal one side, David, organised by one from Edinburgh.

343
00:37:09,430 --> 00:37:17,500
I think it was your old labs and we had one vial of equal, one vial of bam, and then you had to map your DNA using this technology.

344
00:37:17,500 --> 00:37:24,880
How far we've come since those days I was I went out for a beer at lunchtime and I came back in and I was shadowing them.

345
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And in fact, I left it all by mistake. And not only did I shut the grid, but shut the whole apparatus,

346
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and I panicked because I thought I was going to get the sack and I spent the whole hour rubbing the palladium off the side of the tube.

347
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But that was there was different days. It was a fantastic experience.

348
00:37:43,620 --> 00:37:49,800
So I'm going to give you a story about that. That was 1972, but I can be interesting.

349
00:37:49,800 --> 00:37:56,760
Lots of diseases. One of them was typhoid disease. Typhoid is still epidemic across the world.

350
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It's still a it's a disease of poverty. It's underreported, but it's still present there.

351
00:38:01,230 --> 00:38:06,180
Probably around 30 million cases of typhoid. It debilitates children, stops them.

352
00:38:06,180 --> 00:38:13,980
Thriving is the persistent nasty illness which I've studied.

353
00:38:13,980 --> 00:38:18,720
Now, the organism that causes it is is called the bite caused by a bacteria called Samuel Typekit,

354
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and I think about sometimes it's very close to coli.

355
00:38:21,160 --> 00:38:28,050
The genetics are very similar, but it's really unusual in this organism will only cause infections in human beings.

356
00:38:28,050 --> 00:38:35,730
It does not cause infections in any other animals. Its human restricted and I became fascinated by this organism of the is.

357
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It's part this is a genetic landscape over here. If the organism. And it's a tiny part of the salmonella altogether.

358
00:38:44,470 --> 00:38:51,780
So this is a genetic scale of Typekit compared to the whole of the sommeliers typhimurium over here in this power type of a tiny organism.

359
00:38:51,780 --> 00:38:55,140
It entered the human population once from an animal,

360
00:38:55,140 --> 00:39:01,450
and the organism is persisted in the human population from a single entry into the population several thousand years ago.

361
00:39:01,450 --> 00:39:06,330
So all salmonella Typekit have a common ancestor, a single origin bit like COVID.

362
00:39:06,330 --> 00:39:10,950
It's almost. And this has been repeated. All diseases, whooping cough.

363
00:39:10,950 --> 00:39:18,460
Many diseases are of this type where their money for that kind of come into the human population wants.

364
00:39:18,460 --> 00:39:23,320
Just recently, dishes and clubs are very famous for these work on the Kobe Boxing Blondie and myself,

365
00:39:23,320 --> 00:39:31,240
we set up this human challenge study in an Oxford actually, where we challenge people and define the disease typhoid.

366
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And it's this is they are going to stay in the body is an oxygen, enters the blood and persists over many, many weeks.

367
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And if it's not treated, this will continue after many months of infection, it carries a chronic infection and a carrier strike.

368
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It's a nasty fever, and the infectious dose is very low, around 10 to the full 10 to three organisms.

369
00:39:52,010 --> 00:40:01,080
So we work with in the cell three environment is pretty nasty organism. So the time I was doing my stay, there were sporadic reports,

370
00:40:01,080 --> 00:40:09,540
I think if you knew these people to put stand would have known them and they they reported an outbreak of a drug resistant typhoid epidemic in Mexico.

371
00:40:09,540 --> 00:40:20,400
That was pretty rare. You know, it was reported as an unusual incident occurring in Mexico, and it was it was associated with an MDR plasmid.

372
00:40:20,400 --> 00:40:25,920
We never actually identified that plasmid, but I'll come back to this diagram in a second.

373
00:40:25,920 --> 00:40:28,470
So let's move on a bit. So let's move on.

374
00:40:28,470 --> 00:40:37,680
Now, 20 years from when I did my DNA, at that point I was I was the chair of the Infection Immunity Panel at the Wellcome Trust,

375
00:40:37,680 --> 00:40:43,380
and I got a grant came across my panel from Nic White in Vietnam,

376
00:40:43,380 --> 00:40:49,890
and I thought, and it was all about typhoid, and I thought I'd been working predominately, you know, thinking around vaccines for typhoid.

377
00:40:49,890 --> 00:40:55,350
I've been thinking about the disease, but it turned out there was a huge tie for that pandemic in Vietnam,

378
00:40:55,350 --> 00:40:58,230
and there was a welcome centre out in Vietnam,

379
00:40:58,230 --> 00:41:07,050
which is here, which is at the time by Nick Day, but eventually was run by this guy, Jeremy Farrar, who's now the director of the Wellcome Trust.

380
00:41:07,050 --> 00:41:13,440
And so I went out and I met Jeremy and these the team we put together and we started to investigate typhoid at that time.

381
00:41:13,440 --> 00:41:20,940
And this is the 1990s and what what was cause, what the MDA, what was called the MDR typhoid?

382
00:41:20,940 --> 00:41:26,430
And it said that, of course, it's a plasmid, it is a particle we can see and well, and it's a multidrug resistant plasmid,

383
00:41:26,430 --> 00:41:32,960
this is an I gross gel that I talked about, and there was another plasmid in here, which is a phage like plasmid.

384
00:41:32,960 --> 00:41:34,730
And of course, there's nothing unusual about that,

385
00:41:34,730 --> 00:41:43,150
but it was it was it was interesting that we could identify and characterise this organism and start to track it.

386
00:41:43,150 --> 00:41:48,490
And then because I was there, I had some implicit welcome.

387
00:41:48,490 --> 00:41:51,670
We actually decided to sequence the genome in the very beginning of genome

388
00:41:51,670 --> 00:41:55,630
sequencing of all is about the fifth or sixth whole genome sequence of an organism.

389
00:41:55,630 --> 00:41:59,920
We actually took that there was some little Typekit from Vietnam.

390
00:41:59,920 --> 00:42:03,910
And then sequenced and published it in Nature and around two thousand.

391
00:42:03,910 --> 00:42:11,590
So this is moving from where I was a student, where I was mapping plasmids with one enzyme using hetero duplex.

392
00:42:11,590 --> 00:42:17,620
20 years later, I was told genome sequencing the organisms that we could imagine that I would have thought about that when I was a page.

393
00:42:17,620 --> 00:42:24,980
They two. But the beauty of it was because I was trained and endowed in molecular biology, and of course,

394
00:42:24,980 --> 00:42:30,350
when I went to Sanger, which I eventually did to run the patent, I knew everything about DNA sequencing.

395
00:42:30,350 --> 00:42:38,180
I knew even when they were inventing the new technologies, the new Celexa sequencing Illumina, I knew all of the technology at all in my brain.

396
00:42:38,180 --> 00:42:48,010
I knew how plasmids replicated and knew how DNA work. It was beautiful and the underpinning of my knowledge base it day.

397
00:42:48,010 --> 00:42:56,790
So it is interesting that the original plasmid that was talked about in 1970 was actually based around a puppy called R27.

398
00:42:56,790 --> 00:43:04,050
Which we are used to use in the lab at that time. Was it just one of those classical resistance plants within the genes mapped over onto the set?

399
00:43:04,050 --> 00:43:10,290
These elements see the resistance genes. But when I got to the sequence out, actually,

400
00:43:10,290 --> 00:43:21,150
it was really interesting that the resistance genes there's a tetracycline resistance of 10 transpose on here to 10 Tet transposon.

401
00:43:21,150 --> 00:43:29,500
Then there's a chloramphenicol transpose on June 10. This is a heavy metal resistance, which the 10 five one I talked about and is trying mated.

402
00:43:29,500 --> 00:43:36,600
And then as ampicillin, it's almost as if you can count when these antibiotics were introduced and you can you can

403
00:43:36,600 --> 00:43:42,030
actually go back and map the point of introduction of these resistance genes into that organism.

404
00:43:42,030 --> 00:43:48,510
And it just corresponds exactly with the introduction about two or three years after the introduction of the antibiotic.

405
00:43:48,510 --> 00:43:53,760
It's loaded with like a biological clock, and that's all occurred in my lifetime.

406
00:43:53,760 --> 00:43:59,580
But as a research researcher working out of Dave's labs and of course, we used all of those in the lab,

407
00:43:59,580 --> 00:44:06,000
we, I think 10 seven, which we were working with, said they read it wasn't the end of Conrad.

408
00:44:06,000 --> 00:44:15,240
Yeah. So all of these mean if Sydney Brenner was on this one. This is Mary Silber in geology with the very first and all of these.

409
00:44:15,240 --> 00:44:20,730
It was Nazi collect Dave show the slide off early on with the tetracycline resistance.

410
00:44:20,730 --> 00:44:27,250
So it was black. A biography of my history on this, on this slide. And Dave's history.

411
00:44:27,250 --> 00:44:33,180
But then I decided about a few years ago that I wanted to see what was happening across the whole world on my father.

412
00:44:33,180 --> 00:44:37,120
You know, I was interested in intervention. I was interested in vaccinology. I wanted to try to stop time.

413
00:44:37,120 --> 00:44:40,600
I tried to intervene when I was interested in developing vaccines.

414
00:44:40,600 --> 00:44:47,440
And I have brilliant students, actually three or four brilliant students, he told who came to me from Australia.

415
00:44:47,440 --> 00:44:54,820
We decided to map the whole take. The all group members said they are all Typekit originate from one spot.

416
00:44:54,820 --> 00:44:59,290
We decided to collect some Typekit across the world and get a snapshot of what

417
00:44:59,290 --> 00:45:05,790
we were facing if we're going to tackle typhoid as a point of intervention.

418
00:45:05,790 --> 00:45:10,780
And so this is a consortium that will tell it I do have is getting people to work together.

419
00:45:10,780 --> 00:45:18,240
I'm quite open in some ways. I'm not the most brilliant scientist in the world, but I have the ability to so that if you like,

420
00:45:18,240 --> 00:45:24,270
organise processes for actually developing larger projects.

421
00:45:24,270 --> 00:45:32,700
And so the outcome of this study, what we did was we actually took around 3000 genomes in secret who the highest,

422
00:45:32,700 --> 00:45:36,150
you know, effort that anybody carried out in the world. They were all collected into the Sanger Institute.

423
00:45:36,150 --> 00:45:40,460
I was had a passage that Sanger we database all of this and did the analysis.

424
00:45:40,460 --> 00:45:46,360
It was the first analysis involved Illumina or five for the classical sequence,

425
00:45:46,360 --> 00:45:50,340
the first paper we published and we identified a mechanism for building phylogenetic trees,

426
00:45:50,340 --> 00:45:54,960
which is now used to cope with the whole technology of actually used to do the

427
00:45:54,960 --> 00:46:00,810
mapping and identifying variants was developed really at the Sanger at that time,

428
00:46:00,810 --> 00:46:05,610
particularly by Cat, who who was a brilliant mathematician.

429
00:46:05,610 --> 00:46:10,560
So what this is the common ancestor of Salmonella Typhi.

430
00:46:10,560 --> 00:46:15,120
I did the first part this where we've actually done with Mark Almond, who again,

431
00:46:15,120 --> 00:46:22,080
who was a brilliant population biologist, much credit him and Brian Spratt, who followed David at Sussex.

432
00:46:22,080 --> 00:46:24,810
We're behind a lot of this thinking.

433
00:46:24,810 --> 00:46:30,240
And we noticed that if you start off at the common origin, you go around here, these are all different parts of the world.

434
00:46:30,240 --> 00:46:34,260
So suddenly this thing here that spins off around here and then goes off like this.

435
00:46:34,260 --> 00:46:38,520
This is a single outbreak that occurred from around 19.

436
00:46:38,520 --> 00:46:43,590
We must do back to 1990, Matthew, back to India in 1990.

437
00:46:43,590 --> 00:46:47,730
And you can see it. It's an outlier. And it turns out this is MDR typhoid.

438
00:46:47,730 --> 00:46:54,810
It's the same. And the strain. It's almost like I said that single entity entry of terminal type into the human population.

439
00:46:54,810 --> 00:47:02,340
There's been a single entry of a promiscuous typhoid India that has taken over the whole world and displaced all other.

440
00:47:02,340 --> 00:47:12,110
The red is the MDR Typekit. We call it eight fifty eight and it's displaced all of the old Typekit that's been around for several thousand years.

441
00:47:12,110 --> 00:47:16,250
And I've seen this again, I said in C. difficile, I've said it in Staphylococcus aureus.

442
00:47:16,250 --> 00:47:17,930
I've seen it in Klebsiella.

443
00:47:17,930 --> 00:47:27,410
But what you see is the replacement by human intervention of the old diseases, by the new diseases which are adapted to human lifestyles.

444
00:47:27,410 --> 00:47:32,510
It's exactly the same thing that's happened with COVID 19, where you have.

445
00:47:32,510 --> 00:47:39,390
But because of the nature of the world now and because the respiratory virus just spreads much, much, much more quickly, it's the same principle.

446
00:47:39,390 --> 00:47:46,670
And there's something unusual about HIV. It's not the fact that it's drug resistant because it can lose drug resistance, and it still persists.

447
00:47:46,670 --> 00:47:51,020
I think it's something to do with a carrier state in humans, a carry state in the intestine,

448
00:47:51,020 --> 00:48:00,110
although we haven't really proven that, but this is a result of a large consortium of people.

449
00:48:00,110 --> 00:48:03,190
So how do you use that kind of data?

450
00:48:03,190 --> 00:48:10,310
Well, one thing happened was that I was I did a lot of work with in Malawi and we have a welcome centre in Malawi.

451
00:48:10,310 --> 00:48:17,970
If you go back to that previous slide, you will notice that I if I can go back.

452
00:48:17,970 --> 00:48:33,010
I do this. So here, notice that what we've been able to do it we tracked so I was in Vietnam in 1990, but we tracked.

453
00:48:33,010 --> 00:48:37,760
I had a colleague, Sam Carrier, who worked in Kilifi and Nairobi.

454
00:48:37,760 --> 00:48:43,100
And Sam was reporting the appearance of MDR Typhoid into into Kenya,

455
00:48:43,100 --> 00:48:50,470
and at that time it was around 1995, 1996, 2005 as well, that there were three introductions.

456
00:48:50,470 --> 00:48:56,770
And we had a centre down in Malawi, and I I asked them if they'd seen typhoid.

457
00:48:56,770 --> 00:49:04,690
And they said, no, we got very rarely got typhoid. Does it really occur in Malawi's below the radar that you're going to get typhoid?

458
00:49:04,690 --> 00:49:08,470
I think you're going to get a sweep of typhoid coming through,

459
00:49:08,470 --> 00:49:16,180
and that was probably around two thousand and thirteen and this is what happened in Malawi in two thousand and thirteen.

460
00:49:16,180 --> 00:49:24,580
This is a huge outbreak of typhoid driven by this MDR fifty eight point five.

461
00:49:24,580 --> 00:49:29,570
Yeah, finished. And this is really, you know, it's really driven.

462
00:49:29,570 --> 00:49:44,490
The whole replaced not only is to replace typhoid in Malawi at low level, it's become epidemic and it's become a huge political problem to deal with.

463
00:49:44,490 --> 00:49:52,620
So typhoid was brought under control in Vietnam in 2000, but it can still persist in many regions of the world.

464
00:49:52,620 --> 00:49:59,940
You know, we still it. But finally, it's interesting that there's been a huge step up now because there's been an extraordinarily

465
00:49:59,940 --> 00:50:05,130
resistant strain of typhoid appeared in Vietnam and MDR strain called XDR extraordinary resistant.

466
00:50:05,130 --> 00:50:11,460
It's spread right across Pakistan, and it's also started to appear on travellers who, for example, we've had cases that broke.

467
00:50:11,460 --> 00:50:18,210
That is a very promiscuous derivative. It turns out it's a derivative of HIV aids.

468
00:50:18,210 --> 00:50:28,490
So we did an analysis of the strain and this was claim here that the analysis. Of this untreatable, very difficult, treatable typhoid.

469
00:50:28,490 --> 00:50:36,310
And it turns out that if you look at the strain. It's got a place within from E.coli, which carries the extended spectrum of people outside,

470
00:50:36,310 --> 00:50:42,580
but on the chromosome, it's got this huge resistance transposon on a plasmid.

471
00:50:42,580 --> 00:50:46,360
It's got a huge resistance transpose. It's got 10 five oh one.

472
00:50:46,360 --> 00:50:52,960
It's got many of the actually the exact sounds poisons that we had back in the day when I met these,

473
00:50:52,960 --> 00:50:55,930
and it's probably got about 50 to 60 different resistance genes.

474
00:50:55,930 --> 00:51:05,770
It's like a battleship of an organism competitor that it is built for modern life and built for resisting any anti microbe way.

475
00:51:05,770 --> 00:51:10,770
But invent new antimicrobials. This paste is ready to deal with the.

476
00:51:10,770 --> 00:51:15,690
But look, we've come a long way from is the maps of the of the of the transposon.

477
00:51:15,690 --> 00:51:21,480
We can go and look at the baby of a coffin, but the last slide I will finish on time.

478
00:51:21,480 --> 00:51:26,700
We're actually now because we've got this, we understand the epidemiology of the disease where there are variants.

479
00:51:26,700 --> 00:51:34,320
And as mentioned, that we started to deliver vaccines. It turns out that we are part of a project that we are also involved in,

480
00:51:34,320 --> 00:51:40,800
which was to develop through welcome support of strata and Typekit, we're able to develop programmes for intervention.

481
00:51:40,800 --> 00:51:47,700
And we were able to get a vaccine out to these XDR typhoid. And it turns out this vaccine is over 90 percent protected.

482
00:51:47,700 --> 00:51:57,490
The Pakistan government did a fantastic job running out. And you can see that's the way we will have to deal with epidemic and diseases in the future.

483
00:51:57,490 --> 00:52:04,660
Rapid action, rapid intervention and hopefully containment of the disease.

484
00:52:04,660 --> 00:52:09,820
So I just want to finish there is a wish to all through my life,

485
00:52:09,820 --> 00:52:15,950
but I want to say that actually underpinning every part of what I've been thinking about was my training with day.

486
00:52:15,950 --> 00:52:23,770
You can see it with it's come through and the work that David does goes far and far beyond the basic science that he covers.

487
00:52:23,770 --> 00:52:38,230
OK, thank you very much. You had on one of your slides that fluoroquinolones for the driver of the spread of this 58 or whatever it was going.

488
00:52:38,230 --> 00:52:44,050
But it's interesting because that's one of the snips of one of the one things that's not mobile is

489
00:52:44,050 --> 00:52:51,700
that is that is that one of the reasons why that's the drive that you think and that it's not a.

490
00:52:51,700 --> 00:52:56,740
Yeah, that's why I think the reason why these plates evolved and are very restricted.

491
00:52:56,740 --> 00:53:01,510
Turns out that that they are actually very well adapted to the main case.

492
00:53:01,510 --> 00:53:06,310
One plasmid sits beautifully in there, but we've seen the Cipro.

493
00:53:06,310 --> 00:53:12,280
It was invented in Birmingham. The first resistance came around ninety five and it's triggered a lot of recent

494
00:53:12,280 --> 00:53:16,000
epidemics that every time the physicians turn to a new drug to tackle it,

495
00:53:16,000 --> 00:53:23,890
they drive the next wave. And really, the key behind control the AMA is just the control and management of antibiotic usage.

496
00:53:23,890 --> 00:53:30,100
And if you stop using some of these other side, it often seems like I'm trying to call the local physicians will use them.

497
00:53:30,100 --> 00:53:36,040
But if you can control the MDR component drops out and you're left with just the Cipro component.

498
00:53:36,040 --> 00:53:42,400
So we have got I think if we revisit it, we'll have more rational ways of controlling asthma in the future,

499
00:53:42,400 --> 00:53:53,020
provided we can get the political will to apply. I probably should know the answer to this, but how how antibiotic resistance.

500
00:53:53,020 --> 00:54:05,410
I mean, what are the the contributions to the there use in animals is that this is really is that really, really a major issue?

501
00:54:05,410 --> 00:54:13,060
So I'll give you an example when we remember this Australian package down there, quite an extra plasmid for the beetle out times.

502
00:54:13,060 --> 00:54:18,400
And it turns out that when we looked at that flap, it's it's it's a very promiscuous plant in the world.

503
00:54:18,400 --> 00:54:24,790
We think it came in from China and there was a recombination in the intestine into 8:58.

504
00:54:24,790 --> 00:54:29,740
It turns out that they've just built a route between China and Pakistan, of course, to the coast.

505
00:54:29,740 --> 00:54:33,430
And I'm pretty sure that that is one of the reasons why we got this idea.

506
00:54:33,430 --> 00:54:39,760
And it's probably the plasmid itself has been nurtured and in animals.

507
00:54:39,760 --> 00:54:42,550
Of course, you wouldn't map it back if you look at the whole organism,

508
00:54:42,550 --> 00:54:47,090
but you look at the plasmid, you can map it back to the origin and Ventnor animals.

509
00:54:47,090 --> 00:54:58,180
I think there's a. Another example comes, yeah, Palestinian, which is pretty much an animal feed again from China.

510
00:54:58,180 --> 00:55:03,760
There stuff was an antibiotic which was admitted to hospital.

511
00:55:03,760 --> 00:55:09,300
I heard the other interesting thing, I think, is that we forget about heavy metal resistance.

512
00:55:09,300 --> 00:55:16,750
That's why I mentioned 10. Five or one could say heavy metals are managed by silver transport systems or antibiotics in bacteria.

513
00:55:16,750 --> 00:55:24,180
I think the selection you notice that the 10 five is right in the middle of the the antibiotic resistance.

514
00:55:24,180 --> 00:55:36,940
I always see that and I think they pollute the role of pollution and driving antibiotic resistance is underappreciated and is investigating.

515
00:55:36,940 --> 00:55:50,860
Yeah, but there's a literature on extension of beneficial commensal bacteria in the gut, but also that they might be offered resistance genes.

516
00:55:50,860 --> 00:56:01,650
What's your view on the prevention of future public health measures are trying to improve on Bakery's microbiota dysbiosis?

517
00:56:01,650 --> 00:56:09,310
Yeah, definitely. So I have this theory, for example, that the new form of typhoid is driven by antibiotic use is look like C. difficile.

518
00:56:09,310 --> 00:56:18,330
You kill it. The it's controlled. It sits with the microbiota, so it says the antibiotic is sensitive, the applause wiped out, it takes off.

519
00:56:18,330 --> 00:56:25,020
And I think that's another underappreciated driver of these clades, which we need to think about.

520
00:56:25,020 --> 00:56:28,920
And I think the idea of replenishing the microbiota because the other thing,

521
00:56:28,920 --> 00:56:33,810
if you look at the microbiota and look at Amarjit it, it's just everywhere.

522
00:56:33,810 --> 00:56:41,830
All organisms are carriers. There's not just the organisms which are being treated. The indirect selection is is critical.

523
00:56:41,830 --> 00:56:51,970
So good morning, everyone, I think like all the other participants here, I'm delighted to be here in person and to celebrate David's career,

524
00:56:51,970 --> 00:57:01,570
amazing career and science, and I wanted to tell you a little bit about my my time with Dave and how I first interacted with Dave.

525
00:57:01,570 --> 00:57:04,000
And like our two preceding speakers,

526
00:57:04,000 --> 00:57:15,010
I was an undergraduate at the University of Sussex and I was very fortunate to have Dave and Neville Simons as professors of microbial genetics,

527
00:57:15,010 --> 00:57:20,680
and I became completely fascinated by transposons and through Dave's lectures.

528
00:57:20,680 --> 00:57:30,040
And this really inspired my interest in DNA gymnastics, which I have pursued through my entire scientific career.

529
00:57:30,040 --> 00:57:36,430
So I, after walking and Dave site as an undergraduate in my senior year.

530
00:57:36,430 --> 00:57:49,810
I then joined the lab as a graduate student in nineteen seventy nine, and I started at Sussex and then moved to Glasgow with Dave in early 1980,

531
00:57:49,810 --> 00:58:00,400
and my thesis research was on a two and three year old base, and so terrifying was all based on defining the site at which acts.

532
00:58:00,400 --> 00:58:08,650
So before we moved to Glasgow, this is a picture of a group of Sussex, but microbial genetics group.

533
00:58:08,650 --> 00:58:14,590
And so I think you can see Dave's very shy, as you know, and his of hiding in the back there.

534
00:58:14,590 --> 00:58:26,230
And of course, the other really important figure for me was Neville Simons, who also was very supportive of of my career and for many years.

535
00:58:26,230 --> 00:58:34,060
And there are a number of people in this picture who moved to Glasgow, including Afrosphere, who is really delighted to see us here today.

536
00:58:34,060 --> 00:58:45,820
And one person who was actually a natural slag I mentioned a little bit is David Leach, who by work is of an intersected with his on multiple levels.

537
00:58:45,820 --> 00:58:56,500
So after completing my PhD in 1982, I moved to the US and I started working on homologous recombination in the buddy system.

538
00:58:56,500 --> 00:59:02,950
And I've continued in that area through my independent career,

539
00:59:02,950 --> 00:59:13,480
and I moved to Columbia University as an assistant professor in 1988 and have stayed there until now until they kicked me out, basically.

540
00:59:13,480 --> 00:59:18,850
And of course, I had the fortune to know Claudia when he was there as the chair of genetics,

541
00:59:18,850 --> 00:59:29,620
which I now have a joint appointment in genetics department. And so I think all of you know of Dave's amazing scientific career.

542
00:59:29,620 --> 00:59:40,960
And although Dave is retiring, we think I think he actually has a new career where it shows and throwing a haggis and so on.

543
00:59:40,960 --> 00:59:50,740
And so this is a movie that Rodney Ralston gave to me, which is from one of the amber workshops, some recombination that was held in Massey Bridge.

544
00:59:50,740 --> 01:00:13,180
So if I can just try and get this started on it, I think that's one of the things that, you know, more haggis throw up.

545
01:00:13,180 --> 01:00:21,850
I think that's the end of the haggis. Well, maybe you don't have a career and haggis stirring, after all.

546
01:00:21,850 --> 01:00:29,620
OK, so one of the things I've actually really enjoyed since I left the house live is the opportunity to interact with him at conferences.

547
01:00:29,620 --> 01:00:38,920
And we've had many fun times at various conferences over the years discussing science and hanging out in the bar and and so on.

548
01:00:38,920 --> 01:00:42,940
So today I'd like to tell you about one of the projects in our lab,

549
01:00:42,940 --> 01:00:50,170
which is on the role of the Emory Lab and Brad 50 complex in a process called DNA and resection.

550
01:00:50,170 --> 01:00:54,580
And I think a lot of the people in this audience work in bacterial systems.

551
01:00:54,580 --> 01:01:03,100
And so just to remind you that Emory, 11, ran 50, the homologues of SBC seed and bacteria.

552
01:01:03,100 --> 01:01:13,290
And this is David Leach, who's at University of Edinburgh, has carried out a lot of the studies on the CD complex and in bacteria.

553
01:01:13,290 --> 01:01:20,450
As B, C, D, as B, C D is very important for resolving palindromes.

554
01:01:20,450 --> 01:01:26,710
It doesn't really have a prominent role in the repair of double strand breaks, but in eukaryotic systems,

555
01:01:26,710 --> 01:01:33,940
this memory level around 50 has evolved into a machine that recognises DNA double strand breaks and is very

556
01:01:33,940 --> 01:01:43,040
important in both the signalling process from double strand breaks and also in the repair of double strand breaks.

557
01:01:43,040 --> 01:01:47,390
And so I just want to give you a quick reminder of DNA double strand,

558
01:01:47,390 --> 01:01:53,510
right repair mechanisms, which is the area where my lab to stay focussed for some time.

559
01:01:53,510 --> 01:02:01,460
So after the formation of a break, the the ends can be joined by a process called non homologous enjoining,

560
01:02:01,460 --> 01:02:05,510
which involves a simple ligation of the DNA ends.

561
01:02:05,510 --> 01:02:12,350
And this is considered to be an error prone mechanism because it can be gain or loss of nucleotides at the ends.

562
01:02:12,350 --> 01:02:19,610
Prior to ligation, then the other pathway for repairing breaks is homologous recombination.

563
01:02:19,610 --> 01:02:28,940
And this requires template synthesis using a homologous Don't-Don't a duplex and this is usually the system chromatin.

564
01:02:28,940 --> 01:02:33,330
And I'm going to go through these various steps and in a few minutes.

565
01:02:33,330 --> 01:02:42,950
And this pathway is considered to be error free, because normally your templating repair isn't synthesis from a sister committed.

566
01:02:42,950 --> 01:02:53,780
So the Emory Levin complex and you carrots and was actually regulated by a number of cofactors and one of them is called SARS two.

567
01:02:53,780 --> 01:03:02,780
And we abbreviate this is the more complex animals the MBA subunit is replaced by MBA one.

568
01:03:02,780 --> 01:03:15,170
And this plays an important role. It's actually recruited to break-ins very rapidly and recruits a nice hotel, one which is an ATM kinase in humans.

569
01:03:15,170 --> 01:03:21,080
And this activates the DNA damage response through a signalling cascade.

570
01:03:21,080 --> 01:03:27,410
But then the MRI's complex is also required for both of these repair mechanisms, and you carry it.

571
01:03:27,410 --> 01:03:36,620
So it actually interacts with the CPU and like a for to promote this non homologous enjoining mechanism.

572
01:03:36,620 --> 01:03:41,810
And it also plays a role in initiating homologous recombination.

573
01:03:41,810 --> 01:03:50,240
And it does this through a process called DNA and resection, where the ends of process to three primes single strand of DNA overhangs.

574
01:03:50,240 --> 01:03:59,280
And in this capacity, it functions with a coke factor called, say, two, which is phosphorylated by cyclin dependent kinase.

575
01:03:59,280 --> 01:04:06,370
And this actually activates the latent and the nucleus activity within Emory 11 subunit.

576
01:04:06,370 --> 01:04:11,440
To actually Nick the dead ends and form this short overhangs,

577
01:04:11,440 --> 01:04:21,430
which are then processed more by two redundant mechanisms one using the zone one x the nucleus and the other using the DNA to enter nucleus,

578
01:04:21,430 --> 01:04:26,410
which works in collaboration with a helicase called SGI Swan.

579
01:04:26,410 --> 01:04:38,680
Together, these mechanisms create long three prime overhangs, which can then be utilised by rackety one, which is the recchi homologue and eukaryotes.

580
01:04:38,680 --> 01:04:46,960
This promotes pairing and strong innovation to initiate DNA synthesis prime from this donor DNA duplex.

581
01:04:46,960 --> 01:04:52,930
And I'm not going to show you any of the later stages of the the reaction of the Rat 51,

582
01:04:52,930 --> 01:04:56,990
because we've been pretty focussed on this under sections that.

583
01:04:56,990 --> 01:05:04,430
And there's a lot of interest in how you regulate going between these two pathways, particularly in mammalian cells,

584
01:05:04,430 --> 01:05:11,450
because the important role of the BRCA1 tumour suppressor plays in actually promoting DNA and resection.

585
01:05:11,450 --> 01:05:15,200
And this is antagonised by a factor of 53 bpm one.

586
01:05:15,200 --> 01:05:18,230
And this regulates interplay between these factors.

587
01:05:18,230 --> 01:05:25,100
It's important for controlling the decision to go into homologous recombination because once you initiate this,

588
01:05:25,100 --> 01:05:34,160
this mechanism through and resection, it's actually very difficult to divert back to the non homologous enjoining pathway.

589
01:05:34,160 --> 01:05:38,810
So we've been very interested in how my ex works,

590
01:05:38,810 --> 01:05:46,940
and so we know that it's activated during the cell cycle through phosphorylation of its cofactors CO2,

591
01:05:46,940 --> 01:05:52,670
and this reaction has been reconstituted in vitro using purified components,

592
01:05:52,670 --> 01:05:59,000
and it's been shown that the memory level actually cleaves internal to the DNA ends and in vitro.

593
01:05:59,000 --> 01:06:03,800
This happens around 15 to 20 nucleotides away from the five prime ends,

594
01:06:03,800 --> 01:06:09,950
and it creates a neck working through the end of nucleus activity of a complex.

595
01:06:09,950 --> 01:06:14,510
And this nick is an entry site for then bi directional resection,

596
01:06:14,510 --> 01:06:21,980
where Emory Levin uses its three primed to five prime excellent nuclease activity to degrade back to Dana ends.

597
01:06:21,980 --> 01:06:29,960
And then further processing of the five prime termination strands is catalysed by either X or one on day two.

598
01:06:29,960 --> 01:06:41,060
So the questions that we've been interested in are primarily how just how does this complex actually recognise dead ends and cells and where does it,

599
01:06:41,060 --> 01:06:46,370
nick? So how how does it actually carry out this incision away from dead ends?

600
01:06:46,370 --> 01:06:51,030
And how far does it? Where does it go? What is it? A car and the chromatin context?

601
01:06:51,030 --> 01:06:59,870
So we know quite a lot from the in vitro walk about where it connects, but where it happens in vivo is not really fully understood.

602
01:06:59,870 --> 01:07:03,110
We are also interested in whether this reaction continues,

603
01:07:03,110 --> 01:07:11,900
whether you just have one cycle of nicking or whether it continues and is more recognition directed by the chromatin environment around breaks.

604
01:07:11,900 --> 01:07:21,400
Because of course, in cells, new cells, we have to deal with nucleosomes that are coating the DNA.

605
01:07:21,400 --> 01:07:30,890
So in order to address these questions, a very talented postdoctoral fellow in the lab decided that he wanted to be able to map more scenic sites.

606
01:07:30,890 --> 01:07:41,230
That single nucleotide resolution and cells and the way we decide to do this is to actually first set up a system

607
01:07:41,230 --> 01:07:47,950
where we could make multiple defined double strand breaks in the yeast genome so we could just irradiate cells.

608
01:07:47,950 --> 01:07:57,370
But the breaks would be formed randomly, so we needed to actually know exactly where breaks are formed in order to map where resection occurs.

609
01:07:57,370 --> 01:08:04,120
And so Robert developed a system where he expressed a bacterial restriction

610
01:08:04,120 --> 01:08:09,460
and a nucleus SRF one which has an eight base pair long recognition sequence.

611
01:08:09,460 --> 01:08:18,370
So this only has 20 natural sites and the entire yeast genome because the yeast genome is somewhat 80 80 rich.

612
01:08:18,370 --> 01:08:25,000
So he had this on an inducible system where we have a transcription factor that is translocated to

613
01:08:25,000 --> 01:08:31,600
the nucleus when we add beta oestradiol to a medium which will induce expression of this nucleus.

614
01:08:31,600 --> 01:08:37,330
And this just shows yeast chromosomes, which are separated on a Postville gel.

615
01:08:37,330 --> 01:08:44,530
So when I was in Dave's slab, of course, it was wonderful that we could actually separate small DNA molecules by agarose gels.

616
01:08:44,530 --> 01:08:49,360
But using Holtville gels, we can actually separate very large DNA molecules.

617
01:08:49,360 --> 01:08:57,580
And you can actually see the complete stereotype of intact use chromosomes by separating them on this gel system.

618
01:08:57,580 --> 01:09:05,650
So they span from the smallest one is around 200 kb and what, 230 kb in length, which is chromosome one?

619
01:09:05,650 --> 01:09:10,330
And so this is before we induce expression of Sarich one.

620
01:09:10,330 --> 01:09:18,340
And you can see the display of the yeast chromosomes based on size and then also induce expression of SRF one.

621
01:09:18,340 --> 01:09:22,510
You can see that nearly always cites a very high efficiency.

622
01:09:22,510 --> 01:09:32,890
And so after four to eight hours, I saw one actually cuts every single site in the yeast genome and you can see these fragments due to the cleavage.

623
01:09:32,890 --> 01:09:38,680
And this is in comparison with this Ikaros plug, which has been treated with the enzyme in vitro.

624
01:09:38,680 --> 01:09:41,900
Just to show up a phone restriction products.

625
01:09:41,900 --> 01:09:49,810
Now this is an odd behaviour on Postville gels, where the bands actually seem to increase in size over time.

626
01:09:49,810 --> 01:09:53,740
And that is because once DNA and resection starts,

627
01:09:53,740 --> 01:10:00,010
the single strand of DNA overhangs from retard the mobility of the chromosomes chromosome fragments,

628
01:10:00,010 --> 01:10:06,130
and so they actually appear to increase in size of obviously not room.

629
01:10:06,130 --> 01:10:10,210
Now, if we look at a chromosome such as 11, which doesn't have an SRF on site,

630
01:10:10,210 --> 01:10:16,060
this stays intact for the entire time, so it's no off target activity for SRF one.

631
01:10:16,060 --> 01:10:21,310
So we know of a position of every single break that's being generated now.

632
01:10:21,310 --> 01:10:27,880
In order to map where Meyerriecks cleaves, we needed to actually suppress the long range resection apparatus.

633
01:10:27,880 --> 01:10:39,040
So we eliminate a long range resection by using a conditional ablation system to eliminate DNA to an SJS one and an X on one node background.

634
01:10:39,040 --> 01:10:42,100
So once we oblate belonged to a long range resection,

635
01:10:42,100 --> 01:10:50,710
it means that the more X nicks that when we map the five prime and here it will correspond to where my ex nicked.

636
01:10:50,710 --> 01:11:01,690
So now, if we remove overhangs using S1 nucleus to blunt the ends, the endpoints will define where mix cleaved the DNA.

637
01:11:01,690 --> 01:11:10,540
Then we can just add on a series of adaptors and then PCR amplified for deep sequencing.

638
01:11:10,540 --> 01:11:13,270
And this method, which is called One Seek,

639
01:11:13,270 --> 01:11:20,720
was actually developed in Scott Teeniest Lab at Sloan-Kettering by actually a former graduate student from my lab at Laney Membertou.

640
01:11:20,720 --> 01:11:27,870
But she did a lot of important work on end resection during her Ph.D. in My Lab.

641
01:11:27,870 --> 01:11:40,360
OK, so just to look very close to a single SRF one cut in the genome before SRF one is induced, of course, we don't see any of these as one Sikri.

642
01:11:40,360 --> 01:11:51,870
So this just shows the scale here of how many raids we obtained at any particular nucleotide after we induce expression of SRF one for just one hour.

643
01:11:51,870 --> 01:12:02,160
You can see that we do see a peak of signal right at the sort of one cut site, so this corresponds to unprocessed double strand breaks.

644
01:12:02,160 --> 01:12:09,000
And then you can now see also the appearance of S1 secretes that are in the flanking DNA.

645
01:12:09,000 --> 01:12:17,020
So these correspond to the positions where Mry 11 is cleaving within the flanking DNA sequences.

646
01:12:17,020 --> 01:12:26,170
Now, if we look at the average S1 sick signal next to own double strand break site, so this is looking at 40 different DNA ends.

647
01:12:26,170 --> 01:12:30,670
We find that the main reception tracked is around 200 nucleotides.

648
01:12:30,670 --> 01:12:43,410
So within an hour of inducing expression of a transcription factor that induces SRF one, Emmerich's is able to claim around 200 nucleotides from ends.

649
01:12:43,410 --> 01:12:50,940
Now, if we look very close to where the STRIP1 cut site is, we can define the minimum resection tract length,

650
01:12:50,940 --> 01:12:56,680
and we find that cleavage occurs around 30 nucleotides away from SRF reform cut site.

651
01:12:56,680 --> 01:13:02,160
So this is the minimum distance at which more ex acts now in experiments.

652
01:13:02,160 --> 01:13:07,830
I'm not going to show this is actually influenced a little bit by this other DNA binding protein called Khouw.

653
01:13:07,830 --> 01:13:14,790
So far removed too from cells, but cleavage is actually occur a little bit closer to ends around 20 nucleotides away,

654
01:13:14,790 --> 01:13:21,490
which is actually a complete match where one has been found in the in vitro experiments.

655
01:13:21,490 --> 01:13:29,890
Now, if we actually monitor this reaction over time, you can see that the signals start spreading into the flanking dynamic,

656
01:13:29,890 --> 01:13:36,370
so we now actually see multiple rounds of cleavages and after four hours we can

657
01:13:36,370 --> 01:13:40,930
actually see where section tracks are more than five hundred nucleotides in length.

658
01:13:40,930 --> 01:13:46,180
And in other experiments, we've shown that this is actually sufficient to drive homologous recombination.

659
01:13:46,180 --> 01:13:51,670
So in fact, you don't need the long range four section apparatus or homology dependent repair.

660
01:13:51,670 --> 01:13:57,230
The tracks produced by these cleavages are sufficient.

661
01:13:57,230 --> 01:14:06,710
So this is consistent with the idea that Emmerich's cleaves at a distance from it, close friends to ex nuclease activity to back to the brake hands.

662
01:14:06,710 --> 01:14:14,240
And then there's a second round of play which and we think this might be promoted by the single strand DNA binding protein,

663
01:14:14,240 --> 01:14:21,470
which has been shown in vitro to stimulate more cleavage on a substrate within three prime overhang.

664
01:14:21,470 --> 01:14:32,980
So you could have multiple rounds of cleavage. So although this data show that MRI can create long single strand DNA overhangs,

665
01:14:32,980 --> 01:14:40,840
it's important to keep in mind that this repetitive nicking might be attenuated when the other nucleus is engaged to Dan.

666
01:14:40,840 --> 01:14:49,520
So whether this extensive resection occurs by Meyerriecks under normal conditions is something that's not entirely clear.

667
01:14:49,520 --> 01:14:54,590
When I won control, that was very important. And this is another thing I learnt from Dave.

668
01:14:54,590 --> 01:15:02,390
It's very important to do controls, so we want you to know that this activity was already due to the memory loss and nuclease activity,

669
01:15:02,390 --> 01:15:12,350
and we were able to incorporate a nuclear dead l'oeil of memory 11 into the strains where we deplete xa1 and SJS DNA too.

670
01:15:12,350 --> 01:15:21,650
And now, even four hours after inducing us our requirement, if we've met where the S1 secrets are, they're actually right at the SRF one cut site.

671
01:15:21,650 --> 01:15:32,150
And if we blow up this region, this is the sort of one cut site you can see that nearly overeats map exactly at the blunt ends of ARE by SRF one.

672
01:15:32,150 --> 01:15:41,970
And this was very gratifying to us, of course, because it told us that all this activity was due to memory, love and activity.

673
01:15:41,970 --> 01:15:49,770
So, as I mentioned, still, December 11 also told us that conditional depletion system was very tight and that no other

674
01:15:49,770 --> 01:15:59,520
nucleases can process double strand breaks and use other than Emory Levin DNA to an excellent.

675
01:15:59,520 --> 01:16:07,190
So one of the things that I hope you noticed is that the pattern of cleavages appears to be non random.

676
01:16:07,190 --> 01:16:11,420
That we see some very strong hot spots for cleavage in the flanking DX9,

677
01:16:11,420 --> 01:16:17,900
so particularly this this location here, so this is a number of raids at that particular nucleotide.

678
01:16:17,900 --> 01:16:25,610
And we performed a biological replicates and found that the patterns were almost super imposible.

679
01:16:25,610 --> 01:16:32,210
So this non-random pattern of cleavage is something intrinsic to the Marek's activity.

680
01:16:32,210 --> 01:16:38,960
And of course, this is the big question that we've been interested in is what drives this non-random cleavage pattern.

681
01:16:38,960 --> 01:16:48,140
And it's been reported many times in the literature that Emmerich's is completely sequence independent, that it doesn't have any sequence specificity.

682
01:16:48,140 --> 01:16:52,580
So we initially focussed on things like the chromatin environment around breaks.

683
01:16:52,580 --> 01:16:56,240
We looked at transcription patterns around breaks.

684
01:16:56,240 --> 01:17:02,570
We even looked at placing a site next to heterochromatin to see if that would influence Emmerich's cleavage.

685
01:17:02,570 --> 01:17:09,620
None of those things really seem to matter. For me, it seems to be pretty clean and pretty much any chromatin context.

686
01:17:09,620 --> 01:17:13,850
So we start looking more carefully at the sequence around the cleavage site.

687
01:17:13,850 --> 01:17:22,580
And so, Robert, retrieve the sequences around these cleavage sites and then scale those with the S1 six four and found

688
01:17:22,580 --> 01:17:31,880
this very distinctive pattern where we saw a strong preference for C three prime to the cleavage site.

689
01:17:31,880 --> 01:17:41,330
And this is in contrast with the average genomic nucleotide content around the cleavage sites, which you can see is rather rich.

690
01:17:41,330 --> 01:17:49,260
So this bias first seemed like it might be important, and we wanted to know whether we could replicate this in vitro and do this.

691
01:17:49,260 --> 01:17:58,190
We collaborated with the Chaker lab, which had reconstituted this nicking reaction with Marek's and say to in vitro,

692
01:17:58,190 --> 01:18:11,330
and this is just a show finger for one of the payten slides were assays where when you incubate and label substrate with my ex and say to you,

693
01:18:11,330 --> 01:18:16,190
see this enter nuclease products which are clearly just around 15 to 20 nucleotides?

694
01:18:16,190 --> 01:18:23,660
Tremendous. So we know that the cleaves in the middle of this fragment in a region of around 50 nucleotides and we designed

695
01:18:23,660 --> 01:18:32,420
substrates which either had all eight or TS in this region or had CS interspersed every seven nucleotides.

696
01:18:32,420 --> 01:18:40,550
And when we monitored cleavage with these different substrates, we saw strong bias for cleavage on the substrate with CS here.

697
01:18:40,550 --> 01:18:49,690
But this bias still is not probably enough to explain this this a rat non-random cleavage pattern.

698
01:18:49,690 --> 01:18:52,300
Suggesting there might be some of feature.

699
01:18:52,300 --> 01:19:03,220
And so one thing that's important to know about memory 11 50 complex that it actually has very low end nucleus activity with double strand of DNA.

700
01:19:03,220 --> 01:19:13,630
And this is stimulated by saying two. So somehow it's cleaving double stranded DNA, even though in vitro its activity.

701
01:19:13,630 --> 01:19:19,870
If you just take that more complex, it cannot clock cut on double strand DNA.

702
01:19:19,870 --> 01:19:25,210
Now, if you take that more complex, it can actually cleave a single strand.

703
01:19:25,210 --> 01:19:29,350
DNA transitions. And this is all data from Patrick Science Lab,

704
01:19:29,350 --> 01:19:36,730
where he met the cleavages on the substrate with a hairpin capped end and found it quickly at this position in this position.

705
01:19:36,730 --> 01:19:49,240
So it's functioning at these junctions, and we also know that it has the more complex has DNA on winding activity that's dependent on the £58.

706
01:19:49,240 --> 01:19:57,520
So we are supposed to see a sequence around the Emirates next site exhibit any unusual mounting profile.

707
01:19:57,520 --> 01:20:01,870
And if we now take the same type of analysis where we look at Albacete,

708
01:20:01,870 --> 01:20:09,640
the mesh of a mounting temperature around over next sites and weigh that with the S1 six, what we find is quite distinctive pattern.

709
01:20:09,640 --> 01:20:13,510
And this is regardless of the size window of that.

710
01:20:13,510 --> 01:20:24,520
We take so either five, 10 or 15 base pairs wherever next site is shown here by the dashed line, and this is the average melting temperature.

711
01:20:24,520 --> 01:20:30,470
We see that this strong bias for a region of low melt ability downstream of the neck.

712
01:20:30,470 --> 01:20:38,860
So we think that the complex is actually there's some local melting of the DNA, which is actually creating the substrate panicking.

713
01:20:38,860 --> 01:20:45,580
And just to remind you that Emery Le1 reactivity is part of a small family of proteins,

714
01:20:45,580 --> 01:20:55,180
and it undergoes large conformation or change when it interacts with DNA and ATP, which moves the memory of a nucleus channel over here.

715
01:20:55,180 --> 01:21:02,830
And we think that this local melting by around 50 in this region, which is driven somewhat by the primary sequence,

716
01:21:02,830 --> 01:21:08,980
actually creates the favourable substrate for Marek's to Emory Levin to cling.

717
01:21:08,980 --> 01:21:18,190
So just to summarise, we found the king and the Shiites 30 base pairs from ends who influences the positions are spreading over time,

718
01:21:18,190 --> 01:21:25,690
suggesting repetitive necking. We see a slight bias. The cleavage by primes will see with a specific local mounting profile.

719
01:21:25,690 --> 01:21:34,680
And although he didn't show this data, this is only mildly impeded by nucleosome occupancy or transcription.

720
01:21:34,680 --> 01:21:42,090
So just want to acknowledge the heavy lifters on this project, and this is a man named Robert Ganago who's standing here.

721
01:21:42,090 --> 01:21:46,680
This is actually Stone King Sculpture Park, which is a little bit north of Manhattan.

722
01:21:46,680 --> 01:21:51,180
And I recommend a visit in the New York area.

723
01:21:51,180 --> 01:22:03,600
This is our first live outing after lockdown. And I also want to thank our collaborators, Giordano and Peter, for doing the in vitro experiments.

724
01:22:03,600 --> 01:22:10,470
And, of course, a giant. Thanks to Dave for setting me on on this course through my scientific career.

725
01:22:10,470 --> 01:22:19,470
Dave's enthusiasm for science is just remarkable. I mean, it just whenever I see him at a conference, his arms up in the air waving around.

726
01:22:19,470 --> 01:22:23,040
I'm going to tell you this and what's going on, what's going on in your lab.

727
01:22:23,040 --> 01:22:28,410
He's always willing to engage in science, and I hope that the time that I've run on my lab,

728
01:22:28,410 --> 01:22:35,190
that I've been able to impart that same enthusiasm that Dave imparted to all of his training.

729
01:22:35,190 --> 01:22:47,040
Thank you. But a few minutes of questions, but I think would get bonus minutes so that actually I put it in again at the end,

730
01:22:47,040 --> 01:22:54,850
so I don't know if there's any way of looping it if anybody wants to see.

731
01:22:54,850 --> 01:23:03,760
It seemed like there was some sort of a symmetry in the two sides of the double strand of this thing so that on one side,

732
01:23:03,760 --> 01:23:14,270
you might get perhaps more spread on some mornings and then on the other one, perhaps a few more concentrated ones.

733
01:23:14,270 --> 01:23:15,640
It was I correct.

734
01:23:15,640 --> 01:23:24,310
And yes, I mean, we have seen that pattern around some bright sites where it seems to be a bias, where we see more necking on one side.

735
01:23:24,310 --> 01:23:25,300
We were initially,

736
01:23:25,300 --> 01:23:33,880
that's why we were sort of initially driven by this idea of transcription and and nuclear some structure influencing the cleavage patterns.

737
01:23:33,880 --> 01:23:37,990
But even though when we initially analysed the data,

738
01:23:37,990 --> 01:23:44,770
it looked as though transcription was influencing the nicking pattern when we actually set out to as say,

739
01:23:44,770 --> 01:23:49,490
well, we had a promoter where we could regulate the level of transcription.

740
01:23:49,490 --> 01:23:55,600
And next to a bright site, we actually didn't really see any correspondence between transcriptional patterns.

741
01:23:55,600 --> 01:23:59,650
So we're not quite sure why we see that asymmetry at some sites.

742
01:23:59,650 --> 01:24:03,310
It's not we don't see that at every site. I guess my question really is,

743
01:24:03,310 --> 01:24:08,050
do you think that correlates in any way with the fact that then you have one of the sites

744
01:24:08,050 --> 01:24:15,100
that essentially becomes the longer rejected invading strand in the downstream pathway?

745
01:24:15,100 --> 01:24:21,760
Right, right? Yes. And we yeah, so we've been eliminated the downstream pathways here.

746
01:24:21,760 --> 01:24:25,630
So all this is due to the Marek's, but it could be a binding.

747
01:24:25,630 --> 01:24:28,900
I mean, it's interesting that that's the nix that we see.

748
01:24:28,900 --> 01:24:38,860
Would you create the perfect binding site for RPA, which actually finds around maximum maximal binding efficiencies at 28 nucleotides?

749
01:24:38,860 --> 01:24:47,190
So in some ways, when you have a snake, you would actually create the perfect binding site.

750
01:24:47,190 --> 01:24:57,860
And a I guess this is two interconnected questions, I guess one is that to what extent the two sides of the break have to be held together?

751
01:24:57,860 --> 01:25:02,680
Right, right. And that's the first part of the question. The second part of the question is.

752
01:25:02,680 --> 01:25:14,080
Everything I've ever heard about more and more EX-, it's a it's a Panopto zone, all of that, but I've never really.

753
01:25:14,080 --> 01:25:18,370
And even despite Carl Peters beautiful work on the on this structure,

754
01:25:18,370 --> 01:25:26,140
I've never really got a good sense of where the eSIMs SNC side of it comes into this whole process.

755
01:25:26,140 --> 01:25:33,590
In principle, our current understanding the symbology, it doesn't really invoke the why,

756
01:25:33,590 --> 01:25:38,170
why it has this extraordinary structure, why it has these coiled coils. Do you have?

757
01:25:38,170 --> 01:25:41,770
What's the thinking currently writes of a cold.

758
01:25:41,770 --> 01:25:51,820
I mean, it seems to be very important and anything you do to perturb the cold calls or the hook, the dimerisation just destroys the nucleus activity.

759
01:25:51,820 --> 01:25:57,280
So it's something about. So from cold pages paper from two years ago,

760
01:25:57,280 --> 01:26:09,010
they showed that when the complex engages DNA or cold calls stiffened up to form like a rock like confirmation, which is thought to trap.

761
01:26:09,010 --> 01:26:17,560
And there's a channel which can accommodate one duplex not too unlike a lot of the other SNC complexes.

762
01:26:17,560 --> 01:26:22,360
And whether that and there are DNA contacts within the core core.

763
01:26:22,360 --> 01:26:31,600
So it seems to be trapping the DNA with between the cold calls and somehow positioning it for the memory lesson and no cleavage.

764
01:26:31,600 --> 01:26:36,160
Because the other thing that's interesting about memory 11 is it actually cleaves. This happened, captain.

765
01:26:36,160 --> 01:26:39,790
And so it has to be able to cleave in a distance from the end.

766
01:26:39,790 --> 01:26:44,620
And that part is not really clear from copy to squawk, but for the cold calls.

767
01:26:44,620 --> 01:26:48,160
And back to this thing about the coordinated cleavages,

768
01:26:48,160 --> 01:26:59,460
there is somewhat firm resonance group showing that if you remove the more complex than you do, see uncoordinated and resections.

769
01:26:59,460 --> 01:27:04,720
So there's something about having the complex there, which is actually coordinating the DNA and processing.

770
01:27:04,720 --> 01:27:11,890
Unfortunately, because all of our work is done on a population basis, we can't really look at the asymmetry of the two ends.

771
01:27:11,890 --> 01:27:16,780
We're always looking at the pull data from the two sides of the break.

772
01:27:16,780 --> 01:27:18,640
So this really needs some imaging.

773
01:27:18,640 --> 01:27:28,270
There's some work going on in Rodney's lab to look at how Marek's interacts with ends and the asymmetry of the processing of DNA ends.

774
01:27:28,270 --> 01:27:33,490
But the role of round 50, and this is not entirely clear.

775
01:27:33,490 --> 01:27:47,200
I mean, you definitely need the team to engage ATP for this confirmation of Switch, and the C2 subunit interacts with 50, not with Mir 11.

776
01:27:47,200 --> 01:27:52,270
And it's thought that it's actually replacing a linker region, Emory Levin,

777
01:27:52,270 --> 01:28:03,340
to activate and help swing around the so that Emory Levin catalytic site is placed on the DNA because Route 50 actually acts as a gate to the DNA.

778
01:28:03,340 --> 01:28:11,590
So Emory and Chevron cannot access the DNA until it undergoes this conformation change and say is thought to be involved in that part.

779
01:28:11,590 --> 01:28:16,870
But it's that it's also binding to the head domain, not super cold calls.

780
01:28:16,870 --> 01:28:29,250
And we're still somewhat of a mystery in this whole issue of tethering, which has been reported by Emory Lab and how that works is not entirely clear.

781
01:28:29,250 --> 01:28:39,027
So there was one final question here when what went to an OK state level right now until.