Evolution of molecular detective.
And after having talked about the broken the Stone Age and then the bronze Bronze Age, I would suggest to go to the Golden Age.
And this is when, of course, David Shearer was invited to invade a new environment,
a new warmer and drier environment compared to Glasgow, of course, and you can see how dry it can be here in Oxford.
And so when I mean, here it was here at the first floor of the why we call the micro unit, which sadly has disappeared since then.
And so that was in 1994.
And so at that time, in 1994, they arrived in Oxford with a population of survivors of the Glasgow or the Bronze Age, I would say.
And so namely, of course, Libya was there and Sean Collins was here today and also Gary Blakely.
But very rapidly during that year, a new generation or a new population of future molecular detectives colonised the Oxford ecosystem.
And so that was the first generation. Then we had the F1 generation, then we had the two, the three, the four and so on, and so many more.
And so I apologise for those we don't have their picture on on this slide, but there are two reasons for that.
The first reason is that I didn't have enough place to put all the people we were in Oxford at the time.
And the second reason is that for some of them, I couldn't find a picture on the web.
And so, so all these people were. So the competition was very tough.
But most of these people where they were successfully selected during that selection and so many of them carried on their career as you know,
and they spread the molecular detective agencies and they with these heritage all over the world.
So they know many, as you may know, Molecular Detective Agency in different countries and all over the world that I would say in India,
in a in Europe, in America and over in a small country that is somewhere in Europe that is called Belgium.
And so this is where I am and to OK.
And so but at the time, already that the people were in Oxford, they were, as I say, quite so successful.
And so they were allowed to colonise a noble niche with much lower, I would say, a much lower fitness coast.
And of course,
you can see the difference here between the old biochemistry department and this marvellous building where we know the new biochemistry department.
Of course, during this time there was science.
And in fact, when they arrived in Oxford, as Marshall mentioned this morning,
the major topic that we were working on was XDR recombination combination and XDR
recombination is in fact a recombination that is carried out by a couple of two protein,
SIRC and zari. And what do these protein they do?
DNA strand exchange reaction, meaning that they cut the DNA strands and then they sort of exchange.
They rewrite the DNA strands at the level of specific sites using a nice choreography, as you can see here, and also in the picture that is above.
And so one intermediate important intermediate in this reaction is, of course,
the Hollywood junction that is at the meeting at the middle of the slides.
And this Holly the junction, as we heard today from Steve,
was in fact the named Holly the junction because it was discovered in 1974 or 64 by Robin Holliday.
When he was working on the homologous recombination.
And here we are, working on site specific recombination, so I propose to call it Charrette Junction.
And in fact, Basharat Junction is in fact very famous in Belgium.
Everybody knows the Sherratt Junction and I would tell you why, because there is a place in Belgium that is called Chirac.
Chirac in that place in that is there is a junction between two major motorways and in fact, way, way, way, way.
It is so famous. It's because every day there is a traffic jam there.
And so we heard about the shattered junction every day.
So, so you recombination a combination as we as well.
I would tell you as different function, I think ethics, we is going to talk about it.
Just ask me. And so the main. Well, the first rule of XIII recombination was discovered.
Rule was to resolve plasmid commerce. And we know we knew and we we worked on that these plasmids are controlled recombination.
That plasmid is controlled by a mechanism that is called topological filter or mechanism that Marshall talked a little bit about this morning.
And so this mechanism was also known since the Bruhn the Bronze Age coming from Glasgow.
But maybe more importantly, we quite rapidly we found out that well, the detective found out not we, but detective found.
If it seems that the chromosome sites for the resolution of chromosome military males.
That is, this is controlled by a fantastic protein that is called F.T Escape.
And in fact, if GSK is not just a protein that acts in XXXII recombination, it's an important cell division protein.
And so this finding was a real turn in the detective career and all the group, I would say, because it brings David and and the agency on a new topic.
And so instead of staying on very, quite small things, small object like plasmid transposons and Trone,
they became interested on what happens on the wall chromosomes in bacteria and and so doing,
because if this case not just a division protein, but it's also a protein that sort of travel along the DNA on the chromosomes.
And so and so then the group became interested in the dynamic of chromosomes in bacteria.
And so we're going to illustrate that first, for example, here it's the ecology cell,
and within the cell there is the DNA, the DNA and the chromosomes and the chromosomes.
The bacterial chromosomes has to pick for the left and right right before and since they've worked on this issue.
We all know that the Leftwich picture is blue and the the right picture is, of course, right.
And so this is inspired.
Some artists, which which I'm going to show you on a video decided to illustrate the issue of the chromosome within the cells.
So I will start the video. Maybe you've seen this video already. So the problem of the chromosome is so you have the box there.
And one of the problem that we have to solve is how to put the chromosome because it's huge compared to the cells within a box within the cell.
But not just that, because the replication the chromosome at some stage will replicate, so you need two chromosomes to be enter into the box.
And so as you can see, this causes several issues that are difficult to address and also and that the group here started to address these
issues and there are different aspect that we're still at the time there is how does the the chromosome coordinate?
Well, know how this the the chromosome?
Undergoes replication stop. So, OK, so well, there is a problem here.
We're. I. I'm back.
It's gone anyway, so my savings don't disappear.
OK, so so so these are issues that we are going to talk about reputation, but also all the chromosome segregates.
And of course, we're going to talk about CCR and all during all these processes is the chromosome repair.
And so for this third session with three people, we are going to talk and the first people,
the first guy that I will ask to come here is a fix bar from the CNRS in France.
We will who talk about XDR recombination and various aspects of it.
So thanks for this implication.
So I guess I don't have much to say because I wanted to make a recap of politics here, but I think you will go to everything.
Um, so I thought I will make a kind of.
And so it's going to be for use in the region and.
So the original picture. It all starts with because of the fact that we have several leprechauns in there as well.
So it started with me, but then the genome is a bigger big secret I picked on.
And so. I came from the east when I arrived in Lebanon, so for me, I mean, the best way to manipulate, to do a cell cycle to it would be the utility.
So basically, you have plenty of origins. You start replicating.
You go fast. And then once you have replicated you, you keep the two crews together with cuisines, because this way you have a problem.
You can always save yourself homologous recombination, so we have miss these four cuisine.
We have Steve Kay, former weekly communication. And you just decide to segregate to separate them just when you are ready to divide.
And now the primary circle of chromosome. That's what I debated. So you what I'm drawing here is that there is no.
Nothing, but if you just try to separate your two chromosome, you have one chance, not of two to get them tangled.
There is no denying there is no distinction, you're just tangle like that.
And so this is risky.
And so maybe that's why bacteria evolve something where they decided to me, we don't want to do these kind of way before.
I mean, it's safer to have just one origin of publication. And as soon as you do replication, you want to separate them.
You don't run the risk of having these kind of tangled up.
OK. And so you have to partition and then you have to separate them, so you condoms the chromosomes with collegiate conventions.
And so in this way, you don't trust this thing. OK, so that's one problem.
So just because of the way the cell cycle is set. There is a problem with archaea, because some archaea have a circular chromosome in three regions.
And so this I don't understand.
I wrote there is a second problem is the fact that DNA is it's a double Alex, so when you're implicated in the body, you have to unwind it.
And so that's what's happening. So you have a little replication for each year.
So I didn't draw the double helix lecture because it's too complicated for me.
In kind of training. That's the point.
And so when you open it, you are positive that you're insurance, they took taken care of by the judges, but Cesar Chavez is not fast enough.
You can have the whole replication fauquier rotating so you get three cats and.
Those precautions taken care of by to perform.
OK, but if Cooper four is not fast enough, then you can get certain names.
So I'm going to to put a few names on those typical things, so I'm going to introduce you.
This is a kind of ketogenic trick trio, Proteobacteria and so the names, I'm going to introduce you.
So because I'm very interested in your career, so your career in the Tree of Gamma, Proteobacteria is very close to equalise.
The closest member I took on as an owl, so I'll group a to consensus.
That's the point. And so what what what I'm going to tell you is that if you look in the in the tree,
there is something which is common to qualifying economies that we have domesticated, omitting the transferase, which is called them.
And we're going to move this is them. And when they domesticated them, they domesticated with it a set of genes which are always associated with them.
And so you have psyche which binds to any immediate DNA and instead of a normal condensing the movie and we have this massive protein.
And so all of those. Always together.
And so now if we put this on top, we have these so is mutilated normally,
but being the following Sunni militants, Typekit Sticky binds to it and it blocks top of my thing.
So you have a kind of state of cohesion. Then to put up the convincing is, maybe it's not the usual type of sequencing and mapping.
This protein is in fact specialised to keep the two region opposite of the original replication together, which is the terms.
A difference important difference between Korea and Nicaragua that Nicolae lost as they lost the partition machinery and equalise getting equal,
I lost as lost people, they kept it. OK, so OK.
So now we go to chromosome dynamics and two.
And so I just drew one single so that if that being the fork when you need to do to repair something is obviously communication.
And so there is only one who did it. So you have the chance you should resolve it to get a crossover.
That's fine. And so you need to solve the.
You need to.
You have a problem only if you are not in the middle of Kosovo, so you solve it if you have the non-member of Kosovo and this is solved by six.
That's what so this is the machinery that burned out.
Explain to you everything. Yeah. And next year.
In fact, the problem is, how do you know that you have a number of Kosovo, you have to count.
I mean, you say you have an even number of Kosovo, I don't I don't want to influence the president.
So the way? So in fact, it doesn't guarantee just the best way to do it is to do it again.
So you do it just before the chromosome one to separate. So you do it.
At the time of television in determining the origin of the origin, and so at a specific site, yeah, OK.
And so this is possible because this machinery is highly regulated and its activity depends on this huge protein,
which is ActionScript U.S. division in which it's assembled into an anointed DNA pump and it can help position the to the to the tell me,
is that the medicine? And then you can activate cell recognition.
And this is very regulated because.
The positioning of the chromosome, the poor genes on the opposite side depend on the machinery.
It depends on the colonisation of the DNA.
The decision to make the cell division at the appropriate position depend on nuclear integration with human,
which is OK and then maintaining that so many swin positions together will
depend on the matching protein and so might be all part of the same set of way.
OK, so that's the original picture whiteboard.
And so now that's the second part of my thought is the use of Excel.
So I told you they are getting names when 2.4, doesn't it fast enough and getting the same, the same thing is the best way you have to.
You want to try to resolve them at the end. And in fact, this year has been shown to be about two decades inmates.
We can resolve dimer, but it can also be getting it on top of that because being one of the proteins have been co-opted,
so they interact directly with folks of the proposal at the site where it needs to at.
And so everything is goes together. OK.
And then there's the self use of HCR, which is mobile elements.
They said, Oh, there's this machinery inside the cell. They are handy.
And so the Glasgow people explain everything about that.
In fact, many of you are using Excel three of the.
And I mean, for these two to listen to you and a class of elements which are integrated mobile elements,
which protects you have to integrate into the deep state of the host.
So Fiji's. So just to tell you a bit more about those elements, I have to go into the detail of it.
So inside the Excel machinery, most back is composed of two proteins mixture of sweet tea and green and mixed,
yielding the following along with wide dynamics and the deep side is composed of a mixture of C binding sites.
And an excel divining site which are slightly different than the central region,
which needs to be at the right distance so that the two proteins can interact together.
And so. Because this is asymmetric, you have to change, in fact, you can make two types of sign ups,
so this is two sides six weeks old bond switched sides, and so neither SLB will contact will.
So you get this sign up for access, different tactics, different tactics you have seen since.
And then the subsequent tactics will be in trance and the cyclic.
Then these sign ups is what we call a we'll call approach these sign ups and all.
You can make these sign ups where exclusive contracts hail B and C and mechanical tactics I've seen since.
And so you have to persuade you can do recognition this way or this way.
But when you take exotically, that's the word from. A lot of work before arriving in the lab.
What you should give them to decide what you can see that he was able to make some money at the junction.
OK, and the junction you have just the exchange of the Chelsea strength here and with the energy,
you get to this to ensure the Chevron junction, sorry.
And but year much prefer to Theresa arbitrary detention than prefers to make it.
And so. Because of this past way, we always hope that whatever you escape will do is OK.
Just make excel, be active so that now we can finish the job.
And in fact. Surprise, surprise. That was unexpected.
In fact, the fastidious, activated but effective leads to the first sign of exchange.
And so now you make a mixture of the charade junction and swords of.
OK, that's the chromosome damage regulation best way. OK.
Mobile element plasmids. Phage I makes.
I mean, they want to use Excel,
but they don't want to depend on if GSK because GSK makes you active only a tiny fraction of the sales cycle, that's a specific position.
When there is said next to this television, Mashele, show me,
many just want to preserve your dominance when you need it and ensure a fish you want to integrate when you until the say you don't,
we don't want to wait. Which can take days in the environment two days before the events.
And so that's. What the plasmid and the phages are using most of them.
And so the plasmid, the involved, these two very nice, beautiful systems,
so they have two core recommendation sites, but they are sea sequences next to this.
So the of sequences which are going by sea factors which force the formation of proteins in China, which is protocol.
And then he tells you he makes the quality dongsheng.
This old gentleman can either be stable, stable,
stabilised so that replication will from this water can recreate Truman or Mills and James Earl and the dying can be shipped back.
Or. They are even less need that force door injunction, and then Ms Excel Rd do the second step and so you directly go to make a donation.
So that's plasmids. So now I'm going to introduce you a few things about your career.
Just to introduce to the integrated mobile elements, so they built your career.
It's a bit like a teaching. Most friends of your career are non-pathogenic the environmental.
There is only two two-syllable of the value Colella, which we can.
We give rise to the pandemics.
And um, and so and on top of that, they need to acquire the Kodaira toxin and the cholera toxin is born on a knife makes the cholera causing phage.
And so these phage integrate into the site of the first chromosome and sometimes the second chromosome didn't tell you.
Cuomo asked, Do you believe it was two chromosome?
Which is kind of interesting because when you start thinking about only chromosome, you can have much more problems.
And so I'm going to just to tell you how City integrates into the sites and I'm going to introduce you a second element,
which is a toxin encrypted phage. So first, you know, we can see that, in fact, that only the creative team phage.
Indeed, you have also the Chelsea phage. You can have virus one.
You can have all the type of page of your favourites and you make arrays so you can integrate one and then the second one and then reintegrate.
And so and so and that's because those elements of the phage. And so that's how it happens.
You have the first Timex, which carries an upside and uses this as a B sides, and so it creates injuries,
and now you have a metal side and then that's also swinging the two integrated element.
The guitar is always the deep side. This is good for the Senate.
This is good for the element, because if you don't free creative side, then you'll say is fix your minus and so it will be lost in the environment.
And then you have the second element that can integrate.
And so if you look at where is the Deep South and the series of the elements, you can see which one was the element integrated for.
So in this case, I drew. She has integrated first and then Korea because that's what we observe in the evolution in the pathogenic strains.
You always have see before you are.
OK, so when we look at it, that's what it takes to have two beautiful eggshell recombination sites.
The problem that the self-recrimination cites as a good model on the sea to the exchange was this.
But it a wrong spacing fights. There's 12 nucleotide instead of six.
So 12 is Alpha Alex term. So basically the two reconvene is instead of facing each other like that, they would be facing original like that.
This is not possible to do anything that the second most as the perfect spacing you can buy.
You can make connexion between the communities, but you have no meraji to do a recommendation.
So that was a problem. And the problem was solved because, in fact, the genome of the phage single stranded DNA in on the genome of the phage,
those two elements are in opposite orientation. And so they form the stem of a hairpin.
And then this time of wrapping, thanks to the second spacing, you have the good spacing.
And here you have the good energy. And so he can do the catalyses.
And so it can catalyse this wheel. Sites with defend and you get into A. You can get.
You can enter into it. The second element I'm going to introduce is to chance the toxin encrypted, which is always integrated, defrosted.
So this element as a perfect spacing, it has a perfect central region, two or two steps of communication.
And so this element uses the device with. OK.
To get. What are you doing made by day and you get 20 attention?
There's a ritual made by Cairo. You don't you don't want to depend on the state.
And so this element. Eyeballs its own Xia activation factor.
OK. It's a different type of factor that can activate Excel.
If this gets a different food, it's a different semi. That's OK.
Now there is a problem with this, which I do not introduce. You just kept it fast.
If you look at the type of TLC and you look at the second flight, so that's not an easy one sight.
If you put all these elements, you can see accepts the binding you predict.
You don't see binding. You put the two of them together.
You don't see any excess. Shall we find that the comparison with when you produce, you get the two C and D bone?
So you have. So this site doesn't make excel, you cannot buy, so you cannot make a sign ups.
You have just one. And so just for some truths kind of intrigued us.
So we wanted to compare the mechanism of activation of a system on this type of site.
And we said, Oh, we are not just going to sell this site, we are just going to study all the possible sites.
Yes. So we took a frontal attack on sites and we just tested them.
So this is just this integrated task.
Exactly. Chelsea cannot be integrated, if you say, but if we take those elements, we can get.
Some of them can be integrated. Twenty five percent of them, if we use exact now, we have definitive rates as well.
As well at the key from issue now is very,
very intuitive and those elements now that was 2.5 years and now twenty five percent of the sequence can be integrated.
So he just showed you. If we look into the detail of the sequence.
So this is a map where I'm showing the city side and you do the sequence as one, two,
three or four different scenes from the episode, I'm just showing them on the left right.
And and so those are the sequence which have one difference to three or four to difference from the episode, but which are close up to this.
And so I think make them feel sick and make them recombine and make them integrate, but still many of them are very badly integrated.
And once you put feel you can make almost insect free online.
Whether it's closer or closer to two to nothing.
OK, so 15 makes the possibility to recombine again sites which have almost no more to do with a true site where we can buy.
So that's just the last thing.
So what I wanted to say about Excel is that it's a highly versatile communication machinery because you can do interval of mechanistic,
you have similar pathway, you can do the best way. Chromosome demoralisation by its kids, you'd see exactly,
but you can also do the sequence where you can stop at the replication stage with Citrix or Cody one, or you can do the 40 plus way.
So in terms of mechanistic, you can do many, many things. It's completely.
And if you look at the sites you can act on. In fact, we can on sites which are very, very well.
So here I just told you that you can put whatever you want here, basically, and it will be able to recombine.
So I told you before that, it is a very stringent, highly regulated, but in fact, you can make it do almost anything you want.
You can make do well. And the only way, I mean.
Maybe I'm wrong, but I think the only type of virus in communities which I know that we're all
able to produce so versatile in some of the sites of the integrase and so and.
If you look in the genetic tree of. So that's three of them.
That's the exterior recommendations. They still deal very, very closely related and so narrow and.
But they are close to the integrase mean the thousand of the communist from sealed people's integrations.
So I just wanted to say that I think we can speculate again in the initially.
A long time ago, there was this type of family of power in their communities,
and they've been used on one side for the integrase where the integral is just recovered
and this silly domain which allowed them to work on those sites and on the other side.
These these three communities evolve to be stringently regulated.
And so one first thing to help regulators to.
Separate into two communities to force the fact of using two different sites,
and so I would say that building was your, you know, one and evolved to block it from acting all the time.
And then on top of that, the regulation is key.
Yeah. So that's just to say that's a right and they sure etc. in the same meeting.
Yeah. So just as a switch, that's the people I enjoy having in the lab or me are a little after.
And also, I had some on top of gen. I had the chance to be sitting next to John Rawls doing a sabbatical and then to each other.
And then I met thousands from place, I'm sure.
Well. And that's the people that are following the trace of Dave trying to talk you more and more, the things were false theories.
If your names are not at the right place because some people have work at different places to say,
you can work on these loans, you just on this one loan this one.
OK, thank you. Thank you. Also, we have time for one question.
Then. So do you know anything about how safety activates sexyold?
Know how it activates excelled, you know what we just know that's exactly on top of activating the Sylvie's making it forming a sign ups.
But the mechanism? We don't know it interacts directly with, but we don't know.
I mean, even if I'm not sure we know it's activated.
But that's a that's a good question.
We hope that discussing with people will get at some points of chromium, which you may need to discuss with Yemen, but that's not the question.
So Vibrio cholerae has two chromosomes, doesn't it?
We have the the earlier telecon about two. And so one of the chromosomes is very accessible to transposition and exchange rate is more stable.
Could these systems be operating designed to to tweak between the two chromosomes, do you think?
Or is that something? In one of chromosome favouring the transposon type integration, the other one or is that not mono?
In fact. I mean, all the variables are to chromosome.
So one is a true chromosome. It's a general chromosome. The second one is the plasmid, which is inside.
So they are called comets because they are important.
And in the videos, so you have the elements on the two chromosome, but in them for your career, for example,
the to be pathogenic, you need the toxin called the ratepayers to a deal and then decide on the Fox chromosome.
It's a big genetic island. And uh, but on the second chromosome, you have a super zero.
But in other species, you have the shift on different forms of sex chromosomes.
So it's not the reason why you have two chromosomes.
Not. Not just to I mean, nobody know why some people have speculated that because you have to chromosome you truly genes,
you can divide faster and the values are very fast.
So maybe you can divide every 10 minutes, but that doesn't fit freely with all the mothers,
so they'll know at the moment there is no good reason to say why they have a second chromosome.
OK, thank you. So the next picture of this afternoon is so Rodrigo Reis llama.
It could come today, so we will have Rodrigo by the bone zoom.
And Rodrigo, this is with David, I think, early 2000 year 2000 and stay here for a postdoc.
And at the end of that, he got a position at McGill University.
And this is where he is and is.
I hope he's going to join us out of that.
Yeah, yeah. So going to talk about replication?
I did it. Yeah, hi.
Yeah, I did change my title. Sorry about that.
I thought it was more appropriate. Forty four for what it was.
What I want to talk about. And well, first, I'm assuming that you can listen to me and that you are looking at the slides.
So I'll just continue. So I first have to thank Lydia and the rest of the people that helped organising the event to invite invite me.
I'm happy to participate in the symposium celebrating Dave and uncertain,
missing all the lunches and dinners and all the good company of the people that are there.
[INAUDIBLE] have to be next time. OK.
Yes, one second. All right. So what I'm I'm going to be telling you about today.
It's a recurring question that I have in my work, which is how during DNA replication,
which implies the separation of the two strands and the synthesis of these of the of two new complementary strands.
How is that the cell managed to prevent the the excessive accumulation of single strand DNA?
This is an important question because single strand DNA is known to be more prone to DNA breaks and has a higher
tendency to accumulate based modifications like domination and therefore leads to a higher mutation rate.
So one aspect that we study in terms of trying to answer understand this question is looking at the dynamics and the architecture of the replication.
And so what I'm going to be telling you about is the these these aspects of the of the eukaryotic replication.
But before starting my scientific the book,
I would like to go back several years and tell you my personal perspective of what
was going on in day five and how when I when I got there and how that has influence,
what what I've done in India in the rest of my career.
So I got there in 2005, and that was a really exciting time, I think.
And Bernard already mentioned a little bit about that.
So at the time, Sinden and Christof had recently published a paper in Gene Sundeep,
where they really started describing how their nuclear ecology it's organised into, like, is really it.
It contains an organisation that is inherited across many generations, and so it's very stable.
So you have the origin in the middle and and the replicators are decides on a lot of questions so that that was completely surprising.
And then they they delve into they went into trying to understand how those was this changing during the cell cycle and so on.
And there were many questions that this opened many questions, some of them.
Another surprising observation was one year after these first publication.
Now, with Sean, where they observed that there was also a particular arrangement between the two sister chromosomes.
So there were many questions that were opened amongst them.
But I find probably most important were what? What is that?
Is structure in this nucleus? What are the components of the process to study our structure in this nucleus?
And how is that DNA segregation is happening? Some of it I was able to participate in answering the question.
Part of the question of segregation with seemed Wang a few years after where we describe the function of the perfor in the process.
Let me see. And then I was also quite involved in studying the role of Mok Be the assembly complex Macbeth,
which was something that was initiated by Christoph and Alicia and was first published in this paper in 2008,
where the word to describe the effect of in the organisation of the chromosome and the effect of losing Macbeth.
And this project was followed on by a few other people that I interacted closely with Marina and being on and
later taken on by angina that that's going to talk after me where she culminating in the eye in two papers.
One. Where we described how the just the expression of mock beef cooked without any DNA replication could restructure the chromosome,
and in a second quarter, we looked at in a second paper where we looked at the structure of memory, remarkably young Stefan.
So in my time, these were people that I interacted a lot with.
But there were many order of wonderful people that generated this amazing,
stimulating environment that I think I have everyone in here that was in indeed life I love at the time.
I I, I guess I excluded a part two students that were but graduates and both Dr. and
and some recurring members such as Marcelo that could come every summer are here.
And so this this was like really a fantastic environment for for ideas and to start working.
And we did have fun. And I have here two examples.
One, the first one is in 2008. This was the first of all UBC tie dinner where you can see they are looking very sharp.
And the second is in one of our Christmas lunch at the anchor, but we went a few times that the same buffet, it was really fun and on.
But in this in this occupation, we all wear these wicked red week.
And I think we all look fabulous. But I want to draw your attention to the style of which Dave wears the week.
I think it's fantastic. Okay.
So it was in this environment with this fantastic group of people that I started trying to
contribute to the understanding of of the of the chromosome structuring and segregation.
And and I chose to focus on the end of replication.
And the idea of these was that I wanted to test whether DNA replication was contributing to segregating the chromosomal loci.
To do this, I studied first studied the the position and the location at which DNA replication was occurring in the cells.
But later on, with Dave's support and encouragement, I started branching out, leaving the chromosome.
The study of the chromosome structure on a side and focussing more on the fundamental aspects of DNA replication.
And this led me to a collaboration of wonderful collaboration with Martinique,
where we described the stoichiometry of the reptilian observing that one of us has three polymerases
or some single molecule studies also included Stephane and I mentioned the MOBI paper with Anja,
and Stefan was an author. And then more later on in my life,
I continued studying aspects of DNA replication and how this has led to the description of the bacterial replication as a very dynamic complex
where the DNA polymerase on order most basically most of the subunits in the reptilian are exchanging are being replaced every few seconds.
We're still trying to understand what what is the biological relevance of this,
but likely has to do with a more efficient the DNA repair and and maybe dealing with excessive super coiling.
So. So then we it's following this same type of questions and ideas, I now try applying this to the less understood repetition in in eukaryotic cells.
So I have the idea of these was to try developing single molecule microscopy to basically,
as I mentioned, try answering like similar questions in this in this system.
And once I'm going back to trying to understand how is that the rep also manages to coordinate the the two activities,
the Healy case and the various sentences and the are the Ruptly.
Some in eukaryotes is particularly interesting because there there is no known
interaction between the large instant polymerase pulled Delta and the helix,
this EMG helix. Hence, this suggests a model were pulled out.
A different copy of Pearl Delta is required every five about every five seconds, every Okazaki fragment.
So, and furthermore, it suggests that for four days,
coordination between DNA synthesis and unwinding to work there has to be like an efficient turnover pulled up.
We would try to test this model and also try to trying to understand older aspects of the eukaryotic replication.
We went on, as I mentioned, to to set up a single molecule microscopy in budding yeast.
This is a summary of what of our particular experimental procedures.
We use scale attack fusions of different reptiles.
Some surviennent we use. We chose scale attack because this allows us to utilise organic flow of force,
which are superior in intensity and and four to stability to fluorescent Brookings.
Then we use an old trick in microscopy,
which is using integration times of 500 milliseconds when looking at single molecules
so that if a molecule is bound to chromatin is not going to be moving that much.
Therefore, we are going to be able to observe them as spots.
But if they are diffusing, then we can see them like all over the place or blurring the intensity they they must.
And so we don't we don't detect them as spots.
We use High-Low or Applicator to increase the signal to noise, and then we use a single two activation events.
So this this later this day that we are using is for Turkey.
Available is coming from the Gemili Farms. So we use a single for two activation event and then we watch over time.
And what we are recording try to figure out. And here it is.
What is the lifetime of these spots? Since the lifetime of this, but once we correct for photo bleaching,
give salts the an estimate of the dwell time of this protein, some chromatin planning the replacement.
So these are examples. This is this is histone H3.
[INAUDIBLE] attack using the protective variable J if i 04:09 that we got from the farms reaffirms the strain.
Mixing also comes from a Tatiana capo was left.
And so you can clearly see most. In this case, it's Keystone.
So most of the proteins are going to be bound to chromatin. You can see a lot of spots.
Occasionally, you can see that there's things moving blur the spots that are of molecules moving very fast.
We know that we are looking at single copies of Ruptly some subunits because the spots that we observe, they they look like single molecules.
They move like single molecule, and they also are sensitive to stress.
And what I mean is that when we look at those spots, we find examples of the spots bleaching in a single step,
which is a golden sand in the single molecule field.
But also, if we look at the distribution of the intensity of these spots, we see unique animal distribution.
Once more are suspected for single molecules. Then when we look at their movement, particularly at the diffusion coefficient on the alpha coefficient,
the anomalous coefficient of these spots, it fits with what has been previously described for chromosomal loci.
You're seeing. The lack operator of race previously in East, finally, if we treat cells with hydroxyurea,
we can observe that the dwell time of the single strand buying protein RPA goes from 19 sorry, from nine to 18 seconds.
Therefore, of reinforcing the idea that nut cells are the replication forks are being sold and cells are the RPA staying longer in there.
So cells are really active in replication.
The cells that we are looking at are really active in replication and and and all this gives us
confidence that we are really looking at the proteins that are participating in direct replication.
A few a couple more things since we wanted to be able to look at 12 times that span from a few seconds to a few minutes,
we we use different intervals between pictures. One second, a second in 20 seconds.
So if we if we use a one second interval, we we just looked at a single imaging plane.
But when we use these are longer range problems.
We we included three imaging planes to minimise the possibility that when we were seeing the disappearance of a spot,
it was because it was getting out of focus. Finally, we also use a PC and a neon green to determine which were the cells that were undergoing space.
So we segment that the nuclei of those face using this label.
From all these type of experiments.
We generated this type of plots where we are looking at the lifetime, the distribution of lifetime of this, both for a for a particular time.
So we need, you know, like in a particular interval, we feed it to a single exponential function and we corrected for bleach for each time.
And from here, we as I mentioned, we generated we obtained the an estimate of the dwell times and we did this for a lot of
different subunits covering the different parts of the reptiles on the CMG helicase,
different polymerases and other components of the reptilian core components of the represen using different intervals.
This is the summary of what we obtained and so reassuringly expectedly,
but reassuringly still, we observed that not the CMG helicase and polyethylene,
which were expected to bind to chromatin for relatively long time,
like several minutes at least, where indeed we found them to be binding for four months.
We also observed again reassuringly, that RPA was in contrast, binding for few seconds.
I suspect it for for the single strand DNA binding protein, which is replaced actively during synthesis of Okazaki fragments.
Now, there were few surprising observations in our data.
The first is that full alpha, which interacts with this stable Syrian city for what we thought is known to interact with City four,
was bound, therefore only 60 seconds. We were expecting it to be stable part of the complex and and it's it has an intermediate and dwell time.
This means that a single copy of Pool Alpha can synthesise few parameters.
Before I refer refer,
before we went forward and try breaking this interaction with CTF four and we use this mutant point mutation that that doesn't bind anymore to 384.
And surprisingly, we also serve that we can still detect relatively long and binding of 40 seconds and not pull off up to Cromarty.
We still this is since we are still in the descriptive part of the of this part of the study,
so we are trying to understand quite what is what is this telling us?
But by far, the most surprising thing was that we observe that pulled out of the lagging strand polymerase
that I was telling you that we were expecting it to be replaced every few seconds,
we observe it to be bound for about five minutes.
So there's a few possible interpretations of these of this long binding upon Delta.
The first is that maybe what we are observing is that the pull there's up,
there's copies of Pearl Delta that are acting out the larkin's drum and the lines drawn and that are replaced every few seconds.
But we have orders that bind somewhere else, maybe on the leading strand or poorer place.
It doesn't really matter. But that that could last for a much longer time.
And that's. OK.
When I ask him, fine. OK, so sorry.
OK, so I'll I sorry, I didn't keep track of the time.
Well, so I'll just I'll just say that.
I'll just mention that that we think that pulled out is interacting directly to the the rest of the reports.
And and that this suggests that other Saudis are trombone model also for
eukaryotic and has implications for fragment maturation or primitive inheritance.
And and we are trying to understand what, what, order, what, what,
what is the biological function of all these and and what other proteins that interact with Iraqis on how how is that they are interacting?
And so. So I mean these these are the people that did the work, particularly knitting Kapadia and and the funding agencies.
And we have an open position for postdocs to continue work this work.
And I just want to finish with thanking Dave again for all his support and encouragement over the years.
And this is another nice picture of him in a black tie.
Thanks. Sorry for going over time. Thank you.
Thank you, Rodrigo. You do you help believe me? Yes.
OK, fine. So thank you for this nice presentation.
I think we are running a little bit out of time, but maybe, maybe there is space for one quick question.
Look. So maybe I had one Rodrigo's still there, though, it's gone.
Yes. OK, so so is it no possibility to have multiple pull delta polymerase on the repeal zone with the data that you've shown?
So no, we don't think so.
And this is because one part of the the things that I didn't have time to to show you is that we we also try estimating what what is the fracture?
What is the copy number of copies of Pearl Delta bound chromatin?
And we obtain a number of a few hundred copies, which is just too close to the number of the replicas of the active reptiles something in the cell?
Yeah. So I think I think our data is suggesting that it's not.
Yeah. Thank you, Rodrigo. We hope to see you soon.
Yeah. Here in Europe, and we wish you the best in my gill.
So the next speaker is going to be Anjana. Yeah.
So is John and Jenna around? Yeah, I'm here.
Online from that. High, Anjana. So I, as I think the the the.
The trajectory of angina is similar to that, the one of Rodrigo, so again, angina or disease with Dave just after, I think we go.
And then she went on, but I don't remember where it was in the states.
I can't remember where. And then she got to a position at the National Centre of Biological Science in Bangalore in India.
And this is from where she's going to talk to us about DNA repair and searching for homology in during homologous recombination.
OK, great. I'm going to quickly share my screen, make sure it's all OK.
Can everyone see my screen? I can't hear you.
OK. OK. Let me dive right in, so first of all, I am so, so sorry, I can't be there in person.
Maybe there will be a time when these are not so difficult to apply for and then we will move around the world much easier.
But for now, I am also really, really glad to be part of this really special day to celebrate this legacy.
And so thank you, Lydia and everyone for having this online offline format.
So many of us can be part of the city.
So I am, as Bernard said, I'm on Janelle, but in writing and I started my lab at NGQDs in Bangalore in December 2016,
and I was in the official lab between 2008 and 2012 as a graduate student that I studied beef and equal chromosome organisation.
And so this is a drawing of an early B B dimer of Dimer that they've had rendered on his favourite black and red notebook.
I'm pretty certain it's still there with him right now.
He must carry one every time he goes to a seminar, either it's on him or under this chair, but I'm pretty certain he's building one so.
And so what I want to do today is talk to you about a story from my own lab looking at the mechanism of bacterial homology, such during recombination.
And the reason why I thought I'd talk about the story is for two reasons.
One is because I think this for me from my side. I think it's truly a homage to Dave because I think our entire approach of trying to
address this question comes from what I have learnt from him and being in his lab.
And so you'll see a lot of signature of Dave in this looking inside cells doing in vivo biochemistry.
Another reason why I thought this was particularly special for me to discuss is because it brings together two parts of science that I think
Dave is super passionate about looking at the combination and also looking at structural maintenance of chromosome family of proteins.
And in this story, both of them end up overlapping together.
So I'm particularly excited to share this with you. Right.
So very quickly.
So in my lab, we are interested broadly in looking at DNA damage response and repair mechanisms to study how these impact genome maintenance,
evolution, neurogenesis and survival. And we've had some beautiful introductions into double strand breaks apart from from several speakers already.
And I'm going to just sort of summarise some key parts so that we can focus on the aspect that I'm interested in talking to you about today.
So this will be able repairing a double strand because by homologous recombination,
using an uncut copy of its DNA as a template for repairs and as illustrated by Steve Olarinde,
et cetera, these are the early steps of recombination are really well characterised.
We know that you need to reset both ends of DNA to reveal a single strand of DNA, and then that single strand of DNA is used for strand invasion.
And then following that recombination and we already heard about the holiday junction.
To understand this part of the combination, and we know that a key protein involved in this process,
both for homology search as well as ground invasion, is a protein called recce in bacteria and its eukaryotic equivalent at 51.
And so seminal work over several years now has established that it is absolutely essential for for recombination.
We know that recce forms a nuclear protein filament on single strand of DNA that's revealed around the Blake site.
But what we don't quite understand as yet is how the spreading nuclear protein filament orchestrates
homology search to enable recombination and then illustrate why this is still an open problem in the field.
So a lot of beautiful in vitro work establish that there are ways in which nuclear
protein filament can move on DNA while engaging in one day sliding into segment hopping,
all while engaging in microbiology sampling, looking for a homologous template, and once it finds its homologous partner, it pays for repair.
But the problem gets a little bit more complex inside the cell because inside the
cell you've got chromosome organisation chromosome compaction in several bacteria.
You also have chromosome tethering of origin regions, for example,
which can limit how much your your broken piece of DNA can move inside the cell to search for its homologous partner.
So this form of constraint sort of led to the idea that maybe you don't search across
the cell randomly and you search more in the vicinity of where the break occurred,
and the search can be controlled than by how the chromosomes are organised or settled inside the cell.
And so the idea then, is that maybe cells generally tend to engage in recombination in the polls that replicate of cohesion period in bacteria,
and that once you segregate longer distances, it's less likely that you'll be able to do efficient homologous recombination.
So really beautiful work by Christian Lesterland when he was in Deep Shut, its lab showed that that may not be the case.
That. You can engage in sales, can engage in long distance search and do the combination in equally.
And this involves the formation of a long recce nuclear power in filament,
a bundle that spans sometimes the entire length of the cell that somehow manages to orchestrate homology.
Such I'm following this in my own postdoc lab with my lab.
I establish that even in colour back so that you have chromosomal tethering,
segregated pieces of DNA can engage in long distance, homology, search and recombination.
So then this still leaves open the question how inside the cell then does a broken piece of DNA sample
several mega bases of DNA and compacted chromosomal environments to find a homologous partner efficiently?
And so this is the question we set out to address in my lab.
How does the recce filament orchestrate homology search and and they use call-back to consensus as a model system because all
back has this very neat genetic trick that allows you to segregate or separate out cells in specific chromosomal content states.
For example, we can separate out these these type of cells with a single mom replicating chromosome.
We call these cuomos, or we can segregate separate out these type of cells.
These three additional cells are two completely replicated and segregated chromosomes.
So now in these cell types, we can ask about recombination or homology search alone without the confounding
effects of overlapping replication cycles or ongoing replication during this process.
So how do we induce a break? We use the IOC. One system to do that means that an IOC one site near the origin of the application and follow backed up,
and we mark the site using an origin proximal marco ibs-c and then be induced to break while inducible expression
of the IOC one enzyme such that we got one of the two chromosome in these cells and one chromosome in the cell.
And using this system, we've extensively characterised the key steps of homologous recombination.
We can now establish this the steps where lekin nucleus and loads at the double strand breaks.
We can then look at homology so specifically and then look at homology pairing, followed by repair events.
And I'm not going to go into these details because these are quite well established already.
What I'm going to do today is focus on the part of homology search and try to understand how these dynamics play out inside the cell.
And I want to highlight already that this work is has been led by one of my first graduate students, an exceptional graduate student of.
And we work very closely with the theoretical physicist on campus mother and Rao.
And I also thank already all funding agencies here, and we have a preprint of this work that was posted just a month ago.
So please don't have a read when you get a chance. So what are the pros doing?
So I decided to ask about the dynamics of recce in these solar cells,
either in these cells with two chromosomes so we can look at search and repair or in cells with just a single chromosome,
so we can look at just the search process alone.
And she specifically looked at two parts of the rocky film, and dynamix one is where is the filament inside the cell?
And second, how long is the filament so we can we can measure whether it's elongated or a short film.
And so what approach does then is to track Ricky when she makes a break in these cells?
So I'm going to show you her a movie of like with the image every one minute.
But this wasn't done experiments where she's image every five seconds at incremental, at increasing intervals as well.
So what are you going to see here is a break that has been made of this cell pole of the cell.
And Ricky has already started sort of sort of started a nucleus of the cell phone and we're going to now track Ricky over time.
And what we find is that when we image Ricky, it actually moves inside the cell.
So it does form long structures, but it's dynamic. It doesn't stay in one position.
It's actually moving in a very directional manner, from one cell phone to the opposite cell phones.
And once it reaches the opposite cell pole, it reverses and then goes back in the same direction.
And this is quite clear now with this very fluorescence profile.
So what you see here is Ricky starting at one cell pole, it moves in a directional manner to the opposite cell Paul turns around,
goes down again and does this iterative cycling multiple times before it finds its homologous partner.
So Frost then characterises this traversal event by defining a reversal as Ricky,
moving from one poll to the other cell phone and asked how long it takes.
And we get, we see that on average, it takes about 300 to 400 seconds for it to go from one poll to the other,
and it takes about six to eight cycles directly, finally finds its homologous partner.
Of course, sometimes the partner can be found in one cycle itself, but on average it gets there by between six to eight cycles.
So then the obvious question now is how is that moving directionally?
Does it know where the homologous partner is? So this was an easy experiment for us to do, as they would say, a Friday afternoon experiment.
She asked, Is that a homologous partner required for this directional movement?
And so she conducted these experiments in the swarm of cells with just a single chromosome now.
And what we find is that even in this case, they can move in a directional manner, and it never stopped.
So when it reaches the opposite cell, Paul, it turns around and just keeps going back and forth because there's no thought,
no threat to the pelvis and it's almost heart breaking, right? Because it just keeps touching.
So clearly a partner attempted is not required for this movement,
and it's something inherent about the property of the likely filament itself that's allowing it to move in this direction.
So what is driving that movement?
Now we know from really nice work done over several years that Recchia itself is unlikely to be a model so it can drive large scale displacements,
but only be a cycle. We can envision in a cycle that allows for smaller growth and shrinkage of the a filament,
but it's hard to see how it moves across long distances inside the cells.
And so we wondered then what is causing this movement? So we did see that wreckage from several structures inside the cell.
It can form a puncture. It can form an elongated filament that's turning at the pole or elongated filament inside the cell also.
And there's no positional or spatial preference for what type of braking you see based on bed in the cell,
so that it does toggle between various structures. But it's not just toggling between various structures during this process, it's also moving.
So something is allowing for wreckage to not just change in its organisation about filament organisation,
but it's also lot something is allowing the key to move inside the cells. And so the question becomes, What is that something?
And we wondered, maybe we're thinking about this in an incomplete manner.
Maybe it's not just working alone and there's something else involved with it is allowing for this movement to occur.
And so what is then that mystery protein that allows for this movement?
So we went after, I would say, my favourite protein.
We went after them SNC protein rich and not much beef in this case.
Second is a repair specific, some key protein in bacteria.
We know that retcon physically interacts with backing from other bacterial systems, and we also know that it is essential for double strand break,
repair and utilise cells knocked out for can sensitivity to blue myosin, for example.
Even in our own setup, when we ask how many cells are able to do long distance search and repair, we find that in the absence of lichen,
we cannot see the bad events in long distance, which suggests then that is essential for long distance search.
So then we ask the question What happens to Lecky Dynamics if you don't have that in the cells?
So it shows that these experiments and I'm going to show you a movie that took every five minutes.
And as we become clear to you why I'm showing you such long time scale movie, she has done shorter time scale movies also.
What we find is that in addition, deletion recce forms at the break site, but it does not move.
You see a nice filament forming, but you don't see long distance reversal inside the cells.
And this gets even more highlighted when you look at the recce fluorescence profile and point you out to the x axis.
It's going to more than 100 minutes, that confirms, but it does not move after forming at the break sites.
And so just to just to highlight again the comparison when you look at several such recce localisation events over the course of imaging,
we do not see any movement at all. But as I showed you in the wild Typekit, you see this very nice movement of Lecky in a directional manner,
from one cell to the other within a matter of a few hundred seconds.
And this holds true irrespective of whether you have a template for repeat or not.
This is again simply a combination of the property of Lecky and retconned together that enables for for this movement to occur.
And so we wonder then what is it about Rackham that is allowing for for the Leket movement, this large scale movement of Lecky inside the cells?
So we went off and sort of ruled out, you know, only early,
though the more obvious suspects supplicant could be affecting the ask does it affect the rate at which nuclear to the black side?
And we found that it does not affect the rate at which technically it's the bleak side. We then ask, does it affect the later recce?
Filament grows once it is formed at a break, and it does not either.
And then we finally ask, does it affect the filament stability of record?
So we know that in cells that don't have recce, cells can undergo something called reckless degradation,
where they degrade extensively or extensively DNA around the black site.
In the absence of Lekin, though,
you don't get extensive dissection to suggest that it is binding just fine at the at the break site and protecting the DNA from excessive degradation.
So we don't doubt out key filament stability as a problem as well.
So here we are, right? So what I've shown you is that you can can localise at a side once there is a double spring break,
but it's the presence of Recon that allows it to do this long distance movement to then do homology, search of homology, search and repair.
And we know that this movement is essential for long distance recombination because in the absence of lichen, you get no repair at all.
So clearly this the step is enabling Ricky to do homology search inside the cell.
And so then what is the dynamic mechanism by which lichen is driving such rocky filament remodelling and translocation in the cell?
And so we were at this point of the project and we were wondering how can we understand the
mechanism better when we started talking to a theoretical physicist on campus modern ro?
What things about cytoskeleton elements? A lot in eukaryotic systems or microtubule dynamics, for example?
And he looked at our data and he said, Look, maybe there's two ways of thinking about it.
One way of thinking about the effect of lichen is while something like motor driven translocation,
it's possible that retcon is a motor that's driving this long-distance movement of Ricky.
But one one prediction of modern urban translocation would be that you would see a static filament moving inside the cell,
and you wouldn't see large fluctuations in filament length as it's moving inside the cell.
But we do see that. So if you look at the changes in filament length, so this is what we call Delta L.
You see that there is lot larger fluctuations in the lengths of Lecky in the wild type cells, but you don't see so much interaction deletion.
So motor driven translocation alone cannot explain the movement that we're observing inside the cell.
So the other way of thinking about it, then, is perhaps it's affecting assembly this assembly,
so maybe that kind of somehow controlling the rate at which it assembled or disassembled
on a single strand of DNA that then perhaps drives movement inside the cell?
And so Mullen looked at these dynamics specifically,
so he mapped out binding UN binding plates of Lekki on single strand of DNA in the presence or absence of action,
and asked Would there be a scenario where one could explain the binding and on binding rates of
Lekki that are dependent on wrecking that allows for net positive movement in one direction?
And he was able to theorise such a scenario based on the fact that we have a stable length of Lekki finally.
So if you look at the mean length of Lekki, it never extends across the whole length of the cell.
It actually has a fixed mean length that is, you know, around 0.2 percent of the wholesale length,
which is about one micron in a three to four micron long cell.
And so based on this, you said, OK, I can explain all of these dynamics with this equation,
and solving this equation gives you a distribution of lengths that you predict for Ricky
inside the cell that very closely match what we see already in our in our experimental setup.
So this suggests that definitely reckoned is affecting the length of Lekki directly.
Another test for whether Akon is affecting the length of Lekki was the turn to the Recon Apes cycle itself.
So we asked whether the ATP cycle plays any role in the Lekki dynamics we observed.
So there can be an assembly protein has concealed carry and what could be residues that participate in ATP binding and hydrolysis.
And so we mutated these residues specifically and ask them, what effects does this have on breaking movement as well as wrecking filament lengths?
And so we made two mutants who made a mutant added to a mutant that is thought to
affect ATP binding and an idoko mutant that is thought to affect ATP hydrolysis,
although not completely, but somewhat.
And in both cases, we got static filaments that does not move inside the cell with the structure of these filaments with the opposite.
So in the ATP binding mutant, yes, we saw longer filaments, but in the hydrolysis mutant, we saw these very tight functional structures.
We do not sample longer filaments inside the cells,
suggesting that the Ricken ATP a cycle is actually affecting how long or small decayed filaments can be.
And given that the filament does not move in either scenario, this length fluctuation is likely important for Ricky to move inside the cell.
Right, of course. I mean, we still can't rule out water driven translocation because they can use an assembly family feuding,
and there is ample evidence now to suggest that some cities do engage in some form of motor driven translocation by a loop exclusion, for example.
So we don't need to do a lot more experiments to test to see whether we can rule in or rule out this.
In fact, it's possible that it's a combination of both, and that's how we're envisioning this whole scenario right now.
So in some ways, to summarise what I've what I've sort of shared with you so far,
I've shown you that when there is a double spend break and pull it back to cells on the call back to chromosome,
it results in the new creation of molecular nuclear power in filament that is quite dynamic inside the cell.
And this dynamic nuclear protein filament undergoes two things it undergoes dynamic remodelling of its architecture,
but it also undergoes iterative movement inside the cell in the direction of metals.
It goes all the poles searching for its homologues partner,
and we like now envision this movement as a form of ligand driven recce motion via controlling how quickly it associates or
dissociate from the DNA substrate in conjunction with retcons sort of moving the DNA substrate itself along in the cell.
And this could be coming by our exclusion as other assembly proteins do,
or even by zipping with with retcon sort of zipping together the single and the
double standard DNA together as it moves inside the cell to do homology search.
So but what does it mean for homology search itself if you can do it this way?
Right. So the idea of having a reception or iteration built into such allows you to search in a more
efficient manner because it allows you to have a fixed position to which you can come back to.
And so now suddenly you are not dependent on on geometry. If you if you're abroad, you can go in, you're in the long axis.
But if you are a circle, you can go in any axis and come back to your fixed point again and then start the process again.
And so this idea of searching is a lot more efficient than random sampling inside the cell.
And it also doesn't depend on your genome getting mobilised.
You just need your broken piece of DNA to move inside the cells and engage in microbiology sampling as it's moving during the search process.
Right. So this is where we are right now, and this is where we want to go next.
We want to try and figure out what is the molecular mechanism by which Lekin is driving the
key filament dynamics and movement and how important is the position of the black side.
And in driving these dynamics are shown you today. OK, so that's the story, and I already thank the people involved,
so I'm going to shift gears a little bit and thank the person we are here to celebrate today.
So I met Dave, but I had come to Oxford for my master's degree and I met him for a master's thesis project.
And this was the first time I I experienced this question.
I had never thought of this problem before that the cell has a lot of DNA and that DNA
has to somehow fit inside the cell in an organised manner and be used by the cells.
And then when they've explained this problem to me and the slide is quite striking, then Donald shared it with Ted, the slide with me.
And this is a slide that Dave has also used several times to try and illustrate the the issue that the cell is dealing with.
How do you take a chromosome and compact a thousand fold to fit inside the cell?
And this was such a fascinating problem, and I am just so grateful that I had the chance to to engage with this problem and try to
understand how cells do chromosome organisation and compact condensation during this process.
I also learnt that it's not just about chromosome organisation, it's also about chromosome segregation.
It's also about maintaining genomes via DNA repair or recombination.
It's also about doing this in conjunction with activities such as transcription and this
sort of holistic picture of of what it means to to maintain chromosomes inside the cell.
Came in Dave's lab for me, and I am so grateful for that and for the people who allowed me to understand that it's not just one problem,
it's actually many problems together and that all of them have to work together
to allow for a cell to then faithfully propagate life over several generations.
And so I want to thank all of these people too far for sort of opening for me,
the the idea and the understanding of what it means to have a chromosome inside of cells.
And thank you, Dave, for for bringing everyone together and for being so enthusiastic and taking us through this journey of the cells chromosome.
In some ways, I want to I want to sort of replace again and say that, you know, many of us, at least me, I can do for myself.
I come to Dave like Ricky without direction, and there's Dave Shadid's,
who comes in like a cool vein and says, I let you search and let you search for.
Don't answer. But I'm going to guide how you search.
And that's so efficient, and it's been such and such a beautiful journey to experience science with this amazing, amazing person.
And I want to also highlight one thing that I think many people sort of touched upon.
But I want to specifically say I think they've had a great way of bringing people together and giving them an
environment where they can be happy to do science and engage in science for the sake of science driven by curiosity,
driven by by the fun of discovery.
And you see such happy faces in all these pictures because I think it's the environment created for all of us to engage together,
to collaborate together and to answer these fundamental questions in biology.
This doesn't stop doing that.
He joined lab meeting with With My Lab last year, and so thank you, Dave, for spreading your enthusiasm to people here as well.
I think that is just so beautiful that you take the time to do that.
And this is a picture of my graduation with my family and extended family at Oxford just before I left for my postdoc with my lab at MIT.
I'll stop here with this picture that my postdoc Asia had taken at a farm summer school in 2019,
where Davis was there in his characteristic form, with his tube around his neck there to explain how topology works.
And he he again said the same thing that he has always said this is his words from an
interview he had done with a phenotype the Oxford University Biochemical Society Journal.
They talked about how important it is to be passionate and just go and answer questions without being afraid.
So keeping your legacy on the I'm sure all of us are going to go ahead and do just that.
Thank you. And at this time, I'll take questions.
Thank you. I, Anjana, can you hear me?
Hey, Dave, I can hear you. Yes. Lovely to hear from you.
Revkin seems to be an outlier in the sense that every other sea protein that I know of works as a complex with accessory proteins, Gleeson's whatever.
Do you have any knowledge or feeling as to whether Revkin is autonomous?
Yeah, I know that's that's a really good question, we don't know,
so we are trying to set up pull downs right now to try and see what else they can associates with.
We know that E! Affects reckons ATPase activity, so it's possible that that accessory role has been taken up by Ricky instead.
But I mean, it's a good question. We can't rule it out unless we try to see what else they can interacts with.
We don't know the answer to that. High end journalist, I'm over on the side here, and I'm sure you seen as Steve Kornacki so very, very high.
Hi, thanks for saying pre-print. Very nice, very provocative data.
And you know, I took a comment and a question.
So the comment is, you know, the one dimensional search, which is really what you're saying, even though it's directional, is very inefficient.
I mean, you even had that nice chromosome spread at the end of your talk and showed how much DNA you'd have to traverse to go across.
I mean, in terms of it being looped and folded, and we heard talks about that.
So following along that track is is it an is a very fast motor, which most species are not?
And again, I know that's controversial too. But but it just doesn't.
I'll say it doesn't make sense to me. So, but you know, it's the data are the data are beautiful.
So the question for you is I was struck by the movements being much like this par ab system.
And I know there's another system behaves like I can't remember which won the pole to pole move and oscillation.
It just almost seems like the PA system.
So I'm trying to figure out. Yes, you're a and observation is indeed correct.
But does is wreck in fact moving along with some other component that's not tracking the DNA,
but rather somehow moving along some cytoskeleton holes or membrane part, something that's not a long, skinny linear track.
So can you can you have you thought about that and just commented on it?
Yeah, no, that that's a really nice questions. I mean,
we thought of in the context of a because there were these mutants that people had isolated in equally showing that it can associated the membrane.
If you make these mutants, you can break membrane association.
These are not cousins of mutants, and we did try to make mutations in similar areas with Becky, but we didn't see any effect on wrecking movement.
So we didn't think of it in the context of that kind is possible.
Maybe like is associating with cytoskeleton element, maybe MRSA or something that then drives that is acting as a trap.
But just to sort of counter your idea of just pure oscillation, you know,
because if it was just oscillation, one would see sort of linear tracts of the rocky filament.
And maybe if there is time for one movie, I'll play a movie.
If that's OK, we have maybe a minute or half a minute. So that's fine.
OK, I'm going to then we'll play a movie before the answer comes OK. All right.
So if you if you see these movies, you can actually see that the filament is changing quite a bit in its in its shape,
and it's possible that it's actually popping across larger segments of DNA.
And so that can allow for for longer movements, right?
And so it may not be just one d sliding.
It could also be hopping across segments of DNA, you know, maybe even domains of DNA, and that allows them for it to jump across a lot more.
Of course, this needs to be tested. I can't we don't know whether this is really the case, but this is a possibility also.
There. High end diner.
And that was that was really fantastic from from your agency, detective agency outpost.
So I was wondering as in there, I can meet and you still got sort of quite extended filament,
yet you said that they're the commodities such as basically not happen anymore.
There's no right recombination happening.
And that's somewhat surprising that eventually you would think maybe of a long time the right segments would come together.
So this is maybe the role of rock n rather to take off the sort of the wrong bits
of the DNA doesn't prevent the random access of other of other segments of DNA,
and that's that can help to to get the right filament off of the wrong place, basically.
Yeah. Yeah, yeah, yeah. That's that's a nice way of thinking about it.
So I want to answer the question in two parts.
The first part is that Ricky never agrees to called back to I can never make the long enough filament that goes from one poll to the other.
The filament is always less than one quarter of the cell.
And so in that sense, for long distance search, the filament would never become long enough to reach the other end, but it can do the best.
So that's perhaps one reason why we don't see the pattern the can deletion at some point, right?
But it's also possible that Rickon is regulating decades sand invasion capability so
that these two pieces of evidence of that are quite interesting and quite far away.
But so one one piece of evidence is that Rick and stimulates decades IDPs cycle.
And then the other piece of evidence separately is that the sand invasion been been decades doing microbiology.
Sampling also stimulates APIs of Ricky. And so it's it's possible that reckoning is bringing together double standard and single strand
of DNA to stimulate strand invasion and that that allows turnover of Ricky more rapidly.
Again, all of this needs to be tested admissible in vitro and they have.
You didn't teach me how to purify protein, so we need to learn that the hard way now.
And John, I came here as lovely talk. Sorry, sorry, you can't be here.
The I had a couple of questions. One is in your system, you're breaking.
You know, when you had to, both the donor and the recipient have both broken potentially is because idea.
And this is a problem with a lot of these systems. The same problem with the whole system was always a sort of criticism of those is that
you really want to break one and not and then have an intact partner to be found.
I don't know whether that you can make any progress on that. My other question is, have you actually been able to image reckon?
And you know, what does it does? Does it track with Rick or is it?
I mean, has that been has that revealed any insight? OK, great.
So for the first question, we maybe have a cheap mic mechanism,
so we essentially only look at cells where one of the two brick markers are lost in cells,
where we lose both the black site markers, we don't regard them because now we know both the sites are broken.
So we only go after the cells with with a single single break, Malcolm lost.
So that's why we are able to look only at the record at single break, followed by recombination events.
But for the second question, we did look at attraction. I mean, these are very preliminary data and we have to analyse them better.
So that can is actually quite dynamic. It always localises with Becky, and there are few rules.
Silicon does not localise onto the break site without wreckage, so it comes after directly and it closes at the beginning.
But then over time, during the search process, you can see looking at various parts of the decay filaments.
Sometimes it's at the back end, sometimes it's at the front end,
and sometimes it's coating the whole of the filament so that these three main categories,
and we don't quite know as yet how each of these are perhaps leading to the other or or what each of these categories mean for Four-decade movement.
So that's something they need to work out now. Keith, thank you very much.
I'm Jenna for this night's a discussion, so.