Hello, everyone.
Welcome to the DNA repair interest group video
conferences. Today we are pleased to feature 2
distinguished speaker, Dr.
Mahina Monster and Dr. Rafael Pavani. Both are the recipients
of the NHF Fellows Award for research act of excellence,
which recognize outstanding scientific research by NIH,
Each speaker will present, about approximately 2025 min, followed
by a QA session after each.
So please submit your questions on via chat box and
we will, address them after each of the presentation.
So today the 1st speaker is Dr. Mahina M who is post our fellow
at the national issues or for environmental health science.
Or an IHS, working under the mentorship of,
Dr. Thomas, in the DNA replication fidelity group.
So within the laboratory of genome integrity and structural
biology. Originally from Bangladesh, she earned her
MBBS degree and the complete the postgraduate training and
Dr. Mouseer Dam pursued a Ph. At Kumamoto University in
Japan where her dissertation focused on the role of
DNA polymerase, Epsilon.
In the progression of prognosis, the the metro cancer. Recurrent
or researcher at the NIHS Center, on understanding
the inner repair pathways particularly in the interplay
She investigated these passwords by analyzing mutational patterns
in engineer the east providing foundational insights into
how cells manage any damage with a broad implications for
Without further ado, Dr.
Thank you for your kind interaction, and I appreciate
the opportunity to share my work with you all today and
I am Mahina and presenting our work from the DNA
And today I will present our research on the whole genome
study of nucleotide extinction repair and translation synthesis
in sacrifices, and these sisters represents our effort
And before diving into the specific software study, let
me provide some background to set the context of our work.
Here we outline the determinants of DNA replication, fidelity
and repair pathway and DNA replication and repair
pathways are vital for preventing mutation and
In the pre-replication states various repair mechanisms like
manipulated exhibition repair, base accession repair help.
Correct any existing DNA damage before replication
begins. And the replication fidelity stays. The.
No, fidelity is maintained through multiple mechanisms
including polymerase selectivity and externicless proofreading.
And the stall replication for can lead to challenges that
require specific responses such as translation DNA synthesis.
And facilitated it fascinated by DNA polymerase data to bypass
this slation. And finally, in the post replication estates,
Missbass repair mechanisms work to correct any errors
And my focus of interest lies in the neglected exhibition
repair and translation synthesis pathway. How primary objective
was to investigate how mutation in any both in linear and TLS
9 7 if mutations.
India, is there a versatile repair mechanism that's
remotely conserved from bacteria to humane, the pathway
involves over 30, 30 proteins in EU carriers and repairs
And this diagram illustrate the coordinated action of over
30 proteins in u carriers like red 23, red 2 and right 14.
That works in any pathway. In the inner pathway
red 14 that that east homologue of human is X.
Pier plays the critical role in verifying DNA damage after
initial recombination by write for write 23 in global
linear or transcription couple in your pathway, then helic
And my focus of interest is red 14 XPA in human omelet
that verifies DNA damage and stabilize the repair complex.
Before I say it in addition to neglected extension repair,
my research also focuses on, translation synthesis.
Polymer is data. Is the key player in translation
DNA synthesis pathway. It is involved in translation
DNA synthesis when replication by another
And it is the complex enzyme made up, several proteins
and rip 3 is the catalytic subunit of polymerase data.
And this figure shows that translation repair process
involved in DNA damage repair during replication in
replication and assistants that process start when the
And in instruction states rep one the submarine of polymerase
data and other translation polymerases are recruited
to the damage site along with the immigrant marker.
In the extension and state polymerase data helps
extend past the damage of geneside allowing
replication to continue.
And this process is part of what's called DNA
damage. Tolerance or translation synthesis which
helps cell cope with DNA damage during replication.
And final step shows that DNA is successfully replicated. Pass
the damage side marked by blue triangle. And polymer is jetta.
Is not only essential in basic cellular responses but also
involved in several important similar processes. Like creating
diversity in immunoglobulin during B cell maturation.
Go, contributing the cancer cell evolution by increasing.
Mutrition, edging process potentially influencing
the cellular sentences.
And this table gives an overview of the various
roll-up polymerase data both in Vivo and Vitro.
Each row leads different type of damage the organism it
affects and what polymerase jet does in response.
This wide range of function highlights polymerit jetters
importance. Not only in helping cell cope with
damage but also in process like evolution and disease.
The error rate of polymerase data is intermediate
between DNA replication and TLS DNA polymerases.
It is 10 to 100 for lace accurate then its fellow
B family member in the DNA replication operators.
But 5 to 34 more accurate than Y family TLS.
And this slide shows the active site of PB family polymerase
is the image highlights key conserve residue near the
active site which are procel for the engines activity
And below we see a comparison table of these conjugacy,
you in B family polymerases including RB 69 and this
position is important across B.
Family polymerases where it is typically occupied
by hydrophobic residue like leucine and methionine.
And my specific focus in this study is on mutation in
polymerase data, L, 9, 7, 9, F.
This mutation chains leucine to filler and at position 9 79.
Why we choose this mutation?
Because losing at position 979 is highly conserved and
critical. Seat in the active site of DNA polymerase jeta.
By replacing it with thinner and a similar but bulkier
hydrophobic Am I not seed and this chains intended
to reduce the engine's fidelity allowing us to study
We work with 4 is a strain, a wild-type, a victory, a
lot variant affecting polymer status function, red 40,
delete, strain that disturb any year and combination
We compare spontaneous mutation rate in wild tab and mutant
stain in reported gene and we found that the mutation
rate in double mutant strain was significantly higher
These are next slides present a visual representation of the
mutation spectrum we observed in the era 3 reporter gene
across our defined state.
The wildebeest train shows a relatively clean spectrum
with few mutations and this primarily simple based
substitution shown in blue.
And the rate 14 delete chain we observed a notable increase in
mutation frequency particularly interesting is the appearance
of more complex in including deletion and it is shown in red.
Are in the Ref 3 LL. Stain shows a distinct pattern
from both wild type and red 14 delayed with a particular
increase in insertion event highlighted with green.
And in double metering strain, we see the significant increase
in all types of mutation, the striking patterns suggest
linear and TLS don't just work independently they
Previously our lab work on reported in which is provided
as a as a snapshot view of polymerase data metogenesis.
Think of it as just taking a picture through a small
window. However, in our current study we have expanded this
dramatically by employing whole genome sequencing, allowing
And before moving into the methodology of our experiment,
I would like to highlight some important primary steps
we took over for our mutation accumulation experiment
To establish a solid baseline, we begin by analyzing data
from previous studies that You use the reporter team.
These analysis allowed us to calculate both the passes
number and the expected mutation rate for each strain like for
a 500 mutation accumulation rate 3 elap needed 160 passes
But, we went through up to 100 passes.
And now let's wipe through our experimental approach.
We designed a comprehensive mutation accumulation study.
Our experimental workflow include 1st reconstructed
and verified DC string using PCR and sequencing and then
strings were passes for mutation accumulation for 100 passes.
We were start passing with single colonies from
Omojegas deployed. Taking 2 single colonies every 48 h.
Freezing samples at key time points. We also checked from
time to time by markers. And did whole genome sequencing at
18 and 50 passes to check the status of mutation accumulation.
And finally, at 100 passes, we did whole genome sequencing
by Let me know and over sake with high coverage sequencing
rigorous mutation calling using mover pipeline then
And this design allowed us to track how meditation
accumulation over time and determine whether the
combination of this pathway defects lead to affects
Now let's move on to the result of our study on this
slide. We are analyzing the mutation rate of forest teams.
The wild-type, the red 14 delete, rate 3 LF and
double METEN, are denoting by different colors.
The bar graph shows the mutation rates for all mutations and
substitution across this state. And it shows both
the red 14 and double-mutin, exhibit higher mutation rate
And this suggests a significant increase in mutation
rate. When both India is disrupted and polymerize
status function is altered.
And in the table below we can see more detail the wild type
chain had a baseline mutation rate of point 2, a mutation
per deca based part generation and in comparison to rate
alemitians significantly delivers mutation rates.
And this slide shows a detailed comparison of mutation rates
of various types of mutation including single-based
perilation and instruction and complex mutation on
SPEAKER. One types, specifically dramatic increase in complex
mutation in double metal, strain indicated by purple.
And this is the most interesting part of our result and tell
us how polymerase data, particularly in a repair
deficacy background, contributes to mutation complexity.
And these slides summarize that. D types of complex mutation
observed acro's define a chain.
We categorize complex mutation into tendon base pair,
double disk per substitution, bifurcated double base pairs
of a state issue and here been extension, local conversion.
And reciprocal repeat expansion contraction. And that table
highlight the counts and mutation ratio for each
straight, notably the double maintain, strain shows a sharp
And this significant rise suggests that both RAT 14
and polymerase data mutation are critically Critical in
preventing, complex mutation.
Now let me summarize the result. Our whole genome analysis
revealed for critical insight resource. First, st mutation
rate, whole genome sequencing of sacrifices services strain with
We will increase mutation rate compared to the wild type.
Next specific mutation that deletion of red 14 laid to
elevates best substitution mutation while rape 3 LL.
Ferant increase complex mutation rate. Next, synergistic
interaction, the combination of rate 14 delete and rate
3 11 showed the highest meditation rate across all
Lastly, complex mutation. Complex mutation particularly
tendon base pair substitutes and bifurcate double-based pairs.
Ser vice, Titian, were significantly
e levated in double maintenance
chain. And this comprehensive picture suggests a crucial
And our this whole genome and TLS pathways in maintaining
genome stability. And our this whole genome analysis across 100
passes serves as a foundational.
And our this whole genome analysis across 100 passives
serves as a foundational study for understanding
polymerstitis meta genesis.
Provide a available reference point for future
research in this feed.
Now I am highlighting the key implications of our findings and
where we are headed next. Our study revealed 3 major insights.
First, st INNER and TLS pathways. Don't just operate
independently, they work together as a partner in
maintaining genomic stability.
Second, when, at 14 is lost, polymerase data becomes more
error prone, suggesting that 14 helps keep polymerase
data activity in check.
Part the synthetic effect we observe between this pathway
have. Important implication for understanding metrogenesis
and cancer development.
Looking ahead, we plan to challenge this pathways
with exogenous DNA damage to further under their cooperative
role in genome production.
And as we look to the future, my resource aspiration extend
beyond art. Polymerance data though classified as a low
fidelity laser studied member compared to other fellow
And with humans inevitable journey into the space
exploration, understanding how these essential
polymers function in space the environment becomes
Just as Mark Whitney's the Martian had added to
Survivor on Mars. Our DNA European mechanism including
polymerase data will also need to function effectively
And before I conclude, I would like to acknowledge
past my deepest thanks to our DNA replication
viability group at Anais, particularly my supervisor Dr.
Tom Kankill our former lab member our number cities for
Harm mentorship special thanks to Scott Lohan for sequence
statical analysis and supporting me throughout the research.
Thank you to all. My lab mates, especially Sarah
Mars, I'm also grateful to our collaborators at AIDS
especially Adam Barkwter, the UNC Chapel Hill high throughout
Former members whose foundational work helped shape
this project and thank you all for your attention. I am
happy to take now question.
Thank you very much for, for the wonderful presentation,
for the audience, if you have any questions, please type in
the, in the, in the chat box.
Also the panelists, if you have any questions, so
please. Come off mute and and ask a question directly.
Oh, we already have one question from, Bruce Temple. What, do
you think that ARE is acting on?
And here is the base devastation is, in, based
substitution is increased in.
Yeah.
And you collaborate a little on how the. In the manian cells,
how TLS cooperates with N.
Sorry, can you? He's taking the question.
If you if you could elaborate on how TLS and N.
Cooperate in mammalian cells.
We checking is.
Human cells.
We checked in in ESL, but we did not take in in, mammalian cell.
We will take it here.
There's 1 more question in the chat. From Wolf Dietrich
here. Very nice presentation.
Did you have a chance to analyze structural variations?
No, I yet not.
I would think to, Analyzed that's that
to have a. Thank you.
Were spontaneous mutations, I gather. I guess it
would. Bruce, was getting at is there any pattern?
In the mutations that you found. That might implicate
some sort of DNA damage, spontaneous DNA damage.
And you're not.
Oh, sorry.
And, and, repeat the equipment.
Okay. And I think was elaborating on what Bruce
might have meant about what lesions, specifically
asking which damage.
We are a second.
In, future, we are taking, the cis partin and UV radiation
we are are trying to test.
Okay.
Well, what you have shown is spontaneous, spontaneous
mutation. Do you have any idea what might be causing
these spontaneous mutations?
Oh.
So such as base. Lots of basis or whatever.
I will think about it.
As continuous.
Okay, I think perhaps in interest of time
we need to move on.
There's 1 more question there.
Oh, okay, sorry.
Yeah, too.
Actually, 2 now. Do you think? Do you think that the
it's riff 3, the, the, the, will affect upcoming DNTP
or DNA binding affinity.
I think.
I have to know these. At this moment I did
not know, I will think.
Okay, and then, Nila Mahashi wants to know why the L. 9
7 9 if shows lower mutation rates compared to wild type.
But this has increased in double mutants.
Yeah, this is the.
Ill-, web 3 is the, actually low militant. And what in the when
the red 14 is a Is, is it, sleep as some, best serviceitation
and make a gap and this gap is a are not, in normal replication
And what this the wild step petition is.
More than the others.
Alright, so for the sake of time, we have to move
on and to, to the next, to the second speaker today.
Doctor, you can stop sharing and, Dr. Pavani, you can
start sharing your slides.
So I will introduce Dr. Pavani, who is a molecular
biologist and dedicate to elucidating complexities of
a genome integrity with a focus on DNA replication.
Repair and the molecular mechanism underlying various
diseases. During his doctoral study, Dr. Pavani completed
the internet internship at the University of attendee at
His PhD work results in several publications and drug discovery.
His PhD work results in several publications and already
earned him the Outstanding Physics Award in the University
of South, It was on 19, Dr.
As a postdoctoral fellow under the mentorship of
Dr. Andrea. On his way.
His research has a significant advance the understanding of
genome instability in human cells and notably he discovered
that the loss of the XL one is synthetically lethal with BRCA
Additionally, Dr. Pavani conducted a highest resolution
analysis of the formation and the fate of collab,
the replication fork.
Revealing that such events can lead to both single, single,
and, and, and, the, the, the, the, You demonstrated that the
magazine involved in repairing this solution differ from
Double stream breaks. So without further ado, Dr.
Yes, we can.
Hi, can you hear me? Is everything? Yeah, nice.
So, I'm Rafael Pavani.
I'm a postdoc at Indonesian's Vi's lab and I want 1st
to thank Karen and Shozang for the invitation.
That's so I can present my work for you guys today. I'm going
to talk about a specific type of DNA devil strand break that
is coupled with replication for.
So, different types of Devil Strand break can happen inside
a cell. And, the, most well -studied one, we'll call
here canonical grades outside the context of replication.
And these breaks are usually produced by external factors
such as ionizing radiation or restriction enzymes. And
they give rise to double -winded double strand breaks.
You can see here left hand and the right hand of a double
strand break. And as I said, this breaks were very well
studied and basically almost everything we know about DNA
However, another type of break can happen and this And it can
happen as replication forks. For example, when a replication
fork fits a lesion called Nick.
Is a single strand break, DNA interruption and one
of the single trends.
And when the fork hits this lesion, which will collapse
the replication fork, you can lose replay some components
such as the CMG helix.
And according to the current models, this will give rise
to a single when the double strength break. So one end
only different from the .
One This is what the current model said, okay? And this
breaks are very important because they are considered the
primary source of the endogenous that was trend breaks actually
they enhance this amount of necks. That will be
unrepaired. So you increase the chances of collapsed forks.
And although this lesions are very important, they are still
not well understood. So, based on the knowledge we know in
the canonical breaks, when you have a double strength break,
Connomial of the Zen joining that is basically like gating
the 2 ends of the double strand break and this can
give rise to small deletions.
But when sales interest phase and move on, they
prefer to use another type of pathway that is called
a model does recombination.
The main difference here is one of the 1st steps. So the
breaks are processed in a process called end reception
where one of the trends is chopped off forming this 3
This process is very accurate and I will talk
in the next lighting more detail about it. But if
this breaks are respected, but HR is not working.
So we have to another, 2 other pathways called
single strand annealing and micromology mediated and
joining they are considered to be alternative pathways.
And, but still they need the 2 wins of a double strength
break. So, I'm all of those recombination pathway
starts when the breaks are respected, as I mentioned,
Once the brakes are respected, a protein co RPA binds to the
single strand DNA overhangs.
And the complex of Braca one, P 2 and BRCA 2, we recruit rad
51 to this single stranded DNA.
Once RAD. 51 is loaded, it will search for the homology
of the sister chromatids.
And, will invade forming this structure. We call D
-loop. And DNA synthesis will start until it reaches the
knowledge with the second end of the break here.
And then the D loop is displaced. The, the, the, and
the repair is now complete.
However, is a little bit different when we think
about breaks associated with replication as the the
literature suggests these are one end that breaks so
And actually the repair of one-ended brakes is not very
well understood. There is a pathway in East called
breaking this replication where the one ended break is
And this, deal loop can synthesize more than
hundreds of KBs and go into the end of the chromosome.
But however this pathway is not a very good idea
to be a primary pathway to repair lesions because it's
a highly mutagenic process.
We've conserved DNA sentences. And, is it still unclear if
this pathway is the primary path we use it to repair
collapsed forks during S phase?
So we thought that there was a need to study this lesion,
especially in mammalian cells, whether we don't have
much knowledge about it.
So we in our lab, we developed a couple of years
ago one technique, that we can, a map double strength
breaks in the genome.
I'm not entering the details because of time, but here's an
example of a double strength break, canonical 1, 2 ends.
And we basically bind adapters to the free ends and after
the processing. We capture this, this, regions where the
break was and then we can have like negative strand reads
So if just for to give you an example here, here
is a let's say this is a double-strand break.
So we'll bind adapter to the left side and to the right side
and the reads will always come from the 5 prime to 3 prime.
So in a canonical double strength break, here is a
case where it's not respected.
You see a sharp peak on the right side with positive
strand reads this. Strange is being sequenced and on
the left side you have the 5 prime to 3 prime by showing
And if the process of reception happens. 3 prime
overhangs are there but during the end sick processing
enzymes will cleave here.
Because we need blunt ends to bind the adapters and the peaks
instead of being sharp they will spread throughout the region.
So this is the pattern of a receptive brick and this
is a pattern of a blunt double strength break.
Then we ask it, okay, can we use this technique to
identify collapsed forks? So, in theory, yes, because here,
for example, is a leading a strand neck, so when a fork
And then, the adapter will bind here to this region and the
reads will come from 5 prime to 3 prime generating a negative
strength reads and if it's respected as I mentioned we will
What about a lagging collapse in a right moving fork? So
here is the same thing. The the fork will hit the lesion, it
will break and then an adapter will bind adapters here to this
So independent of we are if the lesion is on the leading
or the lagging strand we predict that If, fork is
moving to the right, you have negative strength reads.
And if moved to the left, positive trend reads since these
breaks are one-ended breaks. But to distinguish what is
leading and lagging, we need to target uni directional fork.
So we develop a system to study this type of lesions. So 1st
we needed to map the origin of replications in mammalian
cells and we did that by using a technique called EDU SICK.
We are Sells in g 1 and we synchronously release then
in S phase in the presence of EDU and video calling.
Did you is a timid in analog that will be incorporated
into the DNA and a video calling is a polymer.
DNA polymerase inhibitor. So the fido calling treatment
will allow the origins to fire but they don't wellongate so
we can distinguish here this nice peaks and every peak
We select one origin zone. And, here is okay see because another
technique so that the direction of replication is very clear.
So if we put Nick's here in these squares, you will know
exactly that if you put a Nick here on this square, you will
target mainly right moving forks and if you next year will
So if you unique, different strands, you'll be targeting
leading or lagging strands.
And we developed an inducible, system. Just
about DETAIL NICE has 9 NICE to produce this Knicks.
So our experimental setting is basically putting this
Knicks near to the origins that we fire and the forks
will synchronously hit these lesions and then we will analyze
So we get a synchronous sales. We arrest any g 1, add the
docks to induce the niche and then release into S phase.
And when we did that, at the leading breaks, so with the
leading mix will collapse for leading strength breaks.
We see exactly what we predict negative trend reads
in the right moving forks. And positive trend reads
in the left moving forks, which is expected and is very
Showing that's a collapse at 4. We generate one-ended brakes.
However, to our surprise when we did that targeting the lagging
strength, instead of this clear pick one end peak, we see 2
So it seems the lagging brakes generate 2 ended breaks and
this is very surprising because it's different from what
the current model suggests.
And when we zooming at this breaks, as I mentioned, we can
detect with seconds reception.
We can measure the reception length. So reception at
collapsed forks is around 5 KB for both leading
or lagging breaks.
And of course, the 1st question that arrive at is like, where
does the second end of the double strength break at these
lagging breaks come from?
And then we thought about 2 possibilities. One possibility
is you'll have a lagging neck, the fork will collapse.
CMG with the helices will be unloaded and when a
converging fork hits this lesion it will also collapse.
So it's a double collapse and forming this double-ended
double-strand break. I don't have time to show here.
We tested this model. We blocked the conversion for
and we basically see no difference. So we think that
this is not the correct model.
So we are like, okay, there is a second, type of model that
can explain these results.
But this requires that the CMG helix kind of ignores the
lesion on the lagging strand is important to point that the CMG
travels on the leading strand.
So this way, CMG can simply by pass and since the lagging
strands is discontinuous.
It can just continue since the sizing DNA after the break.
And then the replication itself will form this to end
the double strength break.
And can we test that? It's very hard to test this in
Vivo because the lack of reagents for, for the CMGs.
However, our collaborator at Harvard Medical School,
Johannes Walter, He has a very interesting system to
study this type of things.
Because they use channel pose extracts. They are
very active and functional. And they can label
proteins within this tract.
So they hear, for example, label the genes that is part
of the CMG helix and they also label the fan one that
is a protein that travels with the replication forks.
So this way they can measure the CMG progression and also
DNA synthesis. And it's very nice to see that they can
basically see a replication origin in a tetra DNA.
And then we ask then, okay, so can we put Nick's in
the middle of the system?
They say yes, so we asked them to use the NICE CAS 9 DTNA that
we use in our system and targets the leading or the lagging
strand of a replication fork.
So this is the 1st result. When we have a Nick, on the
leading strand here, the CAS.
90 cases in magenta. You can see here the origin just fired
h ere. Can see that CMG progresses
until we reach as the NIC and the chasm and it's stalled
strand it falls off together with the cas line forming
a 1-ended break. However, when they did that in the
lagging strength, they observe a very different outcome.
Instead of the CMG, stalling at the Kas. 9 next site and at
the neck, CMG just bypassed the lesion, ignoring the presence
of the chasm and the neck.
And, this is, will generate this 2 and a double strand
break via replication by pass.
So, and this make a lot of sense with our model. And
not only that, you can see that after CMG bypassed.
There is a gap forming here where, where the, where
the blue collar is. So we interpret this as the breaks
that started to happen.
Are being now respected, consistent with the 2 end break.
We found it in our So basically in this structural part The
leading strand brakes using CAS.
19 A they behave at the current models. Showing a
formation of single when the double strand breaks.
However, if you target the lagging strand and this can
happen maybe in different contexts. Replication can just
bypass this lesion forming a two- double strand break.
So the second part of the talk, we investigated also for
collapse repair. And basically we ask it if we collect.
Samples at later time points. Do we see the brakes being
resolved? And the answer is yes.
Here is wild type cells. Here's a release 6 h. You
see a very nice signal in the legging or leading breaks.
And after, when we collect 12 h time points, we see that
these breaks are largely gone.
And then I said, okay, we can start testing some
factors involved in a double strain brick repair.
And of course, we started with ride 51. So we depleted
route 51 VS IRNA and we perform at our experiment and
very different from what we observed in the wild type cells.
Under route 51 the pollution the brakes are persistent
And not only that, both lagging or leading strengths,
but not only that, the reception increased a lot.
So from 5 KB that I mentioned to you before, now the brakes
show 40 KB of reception.
So The lack of red 51 generates hyper recepted and unrepaired
breaks. And what is very curious is when you look at the leading
breaks that in wild type are mainly one-ended breaks at
I will get back to this in a minute. And of course, we
needed to test if BIR is really a thing at Collapsed Forks.
And when we did, when we depleted factors involved in
BIR, such as POLY TREE or RAD.
52, we don't see any effect in the resolution of the
DNA breaks. So we concluded that It's seeing the BIR is
not the primary pathway to repair collapsed forks, but
So let's go back to this very interesting thing that
we found at the leading one -ended breaks that are now
converted to 200 breaks.
How this happened? So to understand better this we did
another experiment that we call a strength specific RPA chip.
So if you remember, while RPA is the protein that binds
to the single trend of DNA.
And we observe it, mainly our pay bound to one
strength, which is consistent with a 1-ended break.
And what is interesting is we here is the next site.
So before the the next site. We can see RPA here that is
consistent with our pay bound to the single stranded DNA
But we also CRP signal after the break. And around 3 KB and
this is although it is small it's very reproducible when
we interpret this as a strand invasion followed by sharp DNA
If this is really straining, and when we deplete route 51,
this signal should be gone.
And that's exactly what happened. You don't see
now like positive strand reads after the break.
However, you see a new signal emerging on the other side of
the brake. And this is totally consistent with what we observe
at the NSIC, that is a two -ended break being formed with
Offer to end the double strength break. And this is
not only RAD 51 deficient cells, but when we do this
experiment in BRCA one deficient cells or in BRCA 2 deficient
So it's HR deficiencies characteristic. One end breaks
are converted to 2 ended breaks.
And of course, we ask it if the conversion forks are responsible
for this conversion, since the lagging as I mentioned to you,
the conversion forks are not responsible for the 200 breaks.
So the way we did this, we, created additional guides
that we call blocking guides.
Oh, not enter in detail here, but here you can see the, we
put this blocking guides here.
And the idea is that they will collapse the other
replication for the converging fork. So in this way you
don't allow the conversion for to heat this lesion.
So basically we have here if you just use the guide lead to
right that is a leading guide y
ou will see this nice 200 breaks in RPA chip as I showed you.
But what happens if we block th
e converging for? The prediction is if the conversion for is
really involved in creating the double when the double
However, if conversion fork is not responsible for the
2 ended breaks, then the signal should be unchanged.
And, we see a huge decrease in the signal and this is
very reproducible. So it seems that the converting
forks are responsible for the creation of a 2 winded breaks.
Leading sites in HR deficient cells. And of course, when,
so when we, route to one recruitment is efficient, the
single and the double stream breaks are converted to to
And another interesting observation, of course, we
did as a control but show at interesting results when
we blocked the converging forks in at one end it
Here if you zoom in you can see the pattern I showed
before. Now this pattern of RPA increases a lot.
And we interpret this as there was a transition for short track
gene conversion, a little bit of synthesis after the break to
a long track gene conversion.
So that is a A pathway that resembles BIR found in East.
So we think that maybe if you block the conversion
fork, now a process such as BIR is activated.
So the summary of this part is if you have a 1 ended break.
If you have a converging fork available, the you have a
strand invasion, a little bit of DNA synthesis, but it seems
To rescue here, it to emerge and a holiday junction
will be formed and resolved later. However, if you don't
have the conversion fork, now it seems that a pathway
We didn't test that yet. But is very different from
what we observed in the presence of, conversion forks.
And if you have a 1 ended break, but in the case you don't have
a strain invasion because HR is deficient then the conversion
fork will hit this lesion and also break creating a double
And what is nice about this is, in the HR deficient tumors,
we, one of the prominent characteristic of these tumors
is micromology mediated and joining scars that basically
So We think that this happened in HR deficient tumors. And also
we, Broca one as I mentioned is one important protein involved
in a model of recombination and play major roles in 2
One is control of end reception and the other is a route 51
recruitment. So we are interested in knowing if, one
contributes to these 2 steps in HR at Collapse and Forks.
One we did and sick, it was very surprising. We don't
see any difference between a break in wild type cells or in
bracket on the fishing cells.
So since the Braca one in this case of collapsed fork
is dispensable for reception.
And then we said, okay, let's get the same guide
RNA and put in our wild type CAS 9, to generate a
canonical double strand break.
And when this happens, the effect of reception is very
clear. So we conclude that one is important for reception at
canonical doubles and breaks, but not at collapsed forks.
However, if we get the later time points, the the BRCA one
the efficient cells indeed show at the fact he repair
the brakes are persistent different from wild type.
And if Broca one doesn't work in the reception control,
why does cells have a defect?
Maybe Broca one is important for RAD. 51 loading.
And indeed, when we did chipsik, we see a huge.
The fact in Rad. 51 loading in Braca one deficient cells. So
BRCA one is important for RAD.
51 loading at collapsed forks and not reception. A protein
that is very important in this Brca one context is 53 BP one
because it's the pollution risk is embryonic letality in mouse.
Rescue Park inhibitor sensitivity and this is
thought to be achieved because of the restoration
of this end reception defect.
But since that collapsed forks, Braca one doesn't play any
role apparently at reception of it's 50 TB one like what is
the road that's 50 trivial one affects the repair of collapse
51, sorry, 50 TB one, in BRCA on the efficient cells
and we did the same essay.
And now the brakes are largely repaired again. So, to 3 VP
one affects the repair somehow.
But how, right? And then when we did ride 51 chips sick
at this BRCA 150 triple on the fission cells, we see
the restoration of route 51 filaments and recruitment.
So it seems that, 53 BP one acts by counteracting RAD.
51 filament formation at collapsed forks and had nothing
to do with the reception as it was described in canonical
So the take home message of my talk is collapsed
forks can generate one or 200 double friend breaks
depending on whether the Knicks on the leading are
All of this recombination is fundamental for repairing
collapsed forks and it seems that the BIR or long track
gene conversion that is the name they use in the.
Sells seems to be an alternative pathway when converging forks
are blocked. And in the absence of HR, even an icon deleting
a strength that in wild type generates one end at breaks can
Another very interesting thing is reception at Collapsed
Forces, Braca One, Independence, Distinct for Braco One Row.
That canonical breaks. And 53 BP and efficiency risk is route
51 recruitment at Collapsed Forks and this is totally
independent of reception.
We we have we dig more into the mechanism but I don't
have time to show here. So here is our paper that was
published couple months ago.
So I'll invite you to read if you're interested in this
topic. And also there are 2 other groups that reach
somehow similar conclusions but independent of us.
The one is in East and another from Lorraine Simon
to Lab and another one. In Harvard, for Ralph Scholuleb,
here in the median cells, so I invite you to also take
And with that I finished my talk so I really thank
Andre Nissan's bike for all the support and mentorship.
And all the members of the lab, especially Vino Tree
party that it was doing this project with me.
Almost since the beginning and very, very helpful. And
these people highlighted here are the people in the
lab that work it in some experiments in the project.
And thanks our collaborators, Johannes Walter, Raj,
Better Chaka and Wei Wu.
And thanks very much for your attention.
Thank you. I thank you, Rafael. What a wonderful
presentation. And I let's move to the, QA session.
We already have a bunch of questions, flowing in. So the
1st question is, is a from Dr.
Huang to single end in the DSP and double-stranded as we have
different repair efficiencies.
Actually no, because the single one that that was turn breaks
a nd double and the devil's friend
breaks they We think they are repairing the same kinetics.
Our results suggest that so somehow after we see the maximum
intensity 6 h after the release, but they largely disappear
in both leading or lagging after 12 h, but they largely
So we think they are basically repaired with similar kinetics.
Okay, okay, this question from Michael Lifton. In the fork
Brock experiment the fork blocks themselves seem to induce breaks
But they appeared to be 2 ended.
Yep. Right, I think he's talking about this particular slide,
right? And, yes, we target the leading strands, right?
And, yes, we targets the leading strand of the replication
fork 2 with this blocking next strand of the replication
for 2 if this blocking next because we wanted to make sure
And here I need to remind you that this is a HR deficient,
so we expect this 2 ended breaks, right, even here.
But maybe also he's talking here and the explanation for
that, although they target the leading strand of the
replication for Thank you.
We know that the Knicks are not made in all the cells. So for
example, if you have a Nick.
Here, right? If you don't have this initial nick in a cell, but
you have the blocking guides.
Then the fork that will break here will be the 1st fork
another second so this will be a lagging strand break and
not a leading strand break.
So does that make sense? Because not all the Knicks
are efficient. So, 50, we estimate the 50% of
the cells have Knicks.
So some cells we only have the blocking next
but not the initial Nick. And then this fork will be
the one hitting the neck.
But the blocking was very efficient because we see
differences in the 1st break. So there are a lot of cells
that have all of them, but those results when you look
Okay, Michael has a follow-up question. Well, the different
question is, have you looked at the effect of BRCA one
in 53 BP one mutations on resection at DSPs induced
No, we haven't looked at that. It's a very good
idea actually. Just to, we, didn't compare the mutations.
Like canonical that was and breaks with collapsing
forks. But, I think there are other people that did that.
So there is a paper in nature communications. I believe
they did this type of things and they show differences
in the mutation of collapsed forks versus canonical breaks.
I don't remember right now if they specifically showed between
53 VPP and efficiency, BROCOM, and the efficient cells or not.
I need to remind, but some people look at mutations
that collapsed forks or. Canonical double string
breaks but we didn't
Okay, there is another question from Nicolas Hawk.
So, mentioned, very nice talk.
Can you comments on the weather? Depletion for port d
3 or other expected bir lt ggc factor effects the BR like RPA
chip signal beyond brake size.
Yeah, that's a very, very good question. And yes, the answer
is yes. In our supplementary figure, I don't remember
which number, we showed that the if we depleted party 3.
We decrease this, RPA signal after the break. So it seems
the party tree really helps the extension of RPA and the D-loop
extension right and consequently the RPA loading at the brakes.
So yes, this long track gene conversion seems to be
dependent on party tree. I think that's the question.
Okay. Diego Louis Rubiro says very nice presentation. In your
opinion, could tilless enzymes like Real Riv One and Paul Zeta
play a role in converting one -ended DSPs into 2 ended DSPs
Convergence.
I think it might be possible, though we, I don't have a
clear answer for you because The N 6 only captured DA.
Double friend break so My some other things might
happen there but we then we cannot see it So I don't have
an answer for that, but I believe this is a replication
So I think there is a pathway competition at this replication
lesions. So I believe it is very possible that this
protest can play a role, although our results only
If, we didn't play with Rev one. Or was that to make sure
that they contribute somehow.
So it's a thing that we need to test.
Okay, there is another question from Alexandra Nail. Are there
differences in BRCA one post -translational modification
that contribute to this role in reception in canonical
That's also a very good question and I don't know we didn't
explore any post translation or modifications in Brakawa.
We have an hypothesis that if you look at papers that
characterized the proteins that travel with replication forks.
A lot of, proteins such as x, 1, MRE, 11, they travel
with replication forks.
So we think this reception control is independent of Braca
one because the proteins are already there so they are right
away available to do reception.
So you I think Broca the role of Braca one at Canonical Double
Strand breaks is actually the proteins are not there, right?
The brake just happened. So there is a competition to
which pathway to go but when it's coupled with replication
forks a lot of HR proteins are already traveling
So we feel that this is a prior section environment because
it's a pro HR environment since all the proteins are
already concentrated.
In that.
It makes sense. We have one more question in the
chat from Marcela, Hi Rafael, amazing work.
Are you planning to test this technique in TLS knockout cells?
I think it's very similar to Diego, right? Yeah, we
haven't planned to do that, but I think it's a very good
idea since 2 of you asked.
But we are not sure if, as I mentioned, right, using the
techniques we have in the lab and seek, we are not sure
if the depletion of these lesions will be visible.
Right? If something, if there is a consequence. We don't
know if it will be visible in our end sick, but maybe
if you do like amplicon seek or mutation analysis,
Alright, do we have questions from any of the panelists?
I want to comment on these 2 excellent talks and
clearly there people are thinking of combining the
translation synthesis with the, the double scan rate.
So thank you so much. Yes.
Okay, we do have one more question. I think we have
some time for it. Yeah, from Nathan McGill, very sorry
for the mispronunciation.
Could any of the NC Grees be a reverse forked
structure localizing to the reverse branch of the fork?
If a fork re model is the depleted. Smock, smok Al
one, well HTLF, give a different insect read profile.
Perhaps you can read, in the checks. The That's it.
Yeah. These were amazing questions here. Seriously,
amazing questions. And this question totally makes sense.
I am almost asking if Nathan was the reviewer tree, okay, because
they ask that in our manuscripts and We did, HLTF depletion.
So we, we don't think we are identify reverse forks and we
have some reasons for that.
How you are right, the polarity of the signal will be similar.
So we cannot distinguish between a reverse fork.
Or a canon or a doubles can break. However, we
know that route. 51, for example, is important.
For reverse forks to happen is fundamental protein to
reverse replication forks. And instead of decreasing the
signal, actually increase the signal when we deplete route.
51. And the same is applied to HLTF when we do HLTF
efficiency, we don't see any difference in the signal.
So we really don't think and also doesn't explain
why the leading and lagging would be different since the
reverse forks would show.
I think if these were reverse forks, they would show only
one end it breaks and not to this 2 end break button.
So we really think we are looking only at the NA.
Double train breaks here and not reverse forts.
Okay, we have a question from David Orin. Have you tested
the effects of BLM and Vernus, which have been suggested
to have a role in reception?
We haven't tested it was too many things to do it wasn't at
least to you know understand the nucleases But this is for sure.
A future goal to understand the new places that play
a role there at collapse.
Hey, I think, there's no more question. Karen, do
you want to wrap up or?
No, you go ahead.
Okay, so, again, I would like to, thank both of the, presenter
for their wonderful talk.
It's truly appreciated in the from the point of view of
DNA repaired interest group.
And again, we are looking forward for more
interactions and more of your wonderful art, work.
Thank you very much.