From Salem State, the BS Biology and an option in
Biomedical Science at the Chemistry minor in May of 2023.
While an undergraduate, Pamela's biological curiosity was piqued
by numerous courses here.
What further cemented her desire to become a research scientist?
For two summer research experiences, I think it was two of them.
At the Ragon Institute, working with Doctor Schiff Pillai,
I chatted with Pamela's Mama.
Money would go.
Pamela, like me, likes tennis time.
I'm going to the gym, but she's very busy doing her PhD at home.
But you aren't playing as much tennis, I hear.
No, unfortunately not.
Pamela is our PhD candidate at Brandeis University and today she'll tell us about
her research and her talk entitled The Intersection of Homologous Recombination
and Heterochromatin Maintenance in the Sacrifice.
Sarah Vizier.
So welcome back, Pamela.
Thank you so much for that lovely introduction.
So yeah, my name is Pam.
I am in the Haber and Ragunathan labs at Brandeis University,
and I am a second year PhD student in the Molecular and Cell Biology program.
So this festival in in Salem State in general holds a very special place in my
heart.
I made a great community here and I am still in touch and love all of the
professors that taught me a lot of things and sort of paved the way for Graduate
School for me.
So I'm very excited to tell you about my research project and how I'm beginning it.
So I am in both the Haber and Ragunathan labs,
but I'm going to be talking to you all about research mostly in the Haber lab.
So to begin, when we were born, we inherited 23 pairs of chromosomes from
our parents.
And this was arranged in a very specific way.
And despite through going through countless cell divisions,
that arrangement has remained largely stable, ensuring proper development and,
and function.
And so over here, let me put the laser over here,
we're looking at a karyotype or a visual representation of chromosomes where we
can see the number, size and structure.
And here we're looking at a normal individual.
And so this ability of of a cell to maintain its integrity of its genomic
material is, is really important because it prevents
mutations and chromosomal rearrangements.
Now the exception to this, of course, is what happens in tumor cells.
So that stability is disrupted and that leads to a lot of genomic instability,
while cells can develop mutations and chromosomal abnormalities.
So here we're looking at a karyotype of a, of a cancer cell where you can see that
that three distinctions that I told you are very, very different.
You can see that there are a lot more chromosomes, they look very different.
And so a serious threat to genome stability is adna double strand break.
And so in the Haber lab, we are interested in studying DNA double
strand breaks.
And so if we were to zoom into the nucleus of a cell,
we would find this chromosome.
And if we were to decondense it to expose the naked DNA,
we would find this DNA double helical structure over here.
And so a double strand break is just a kind of DNA damage in which both of those
DNA helixes can break.
And when that breaks, that kind of damage can lead to a lot of
serious threats.
And if that isn't correctly repaired, it can lead to mutations and chromosomal
rearrangements.
So understanding how double strand breaks are generated and repaired is really
important to sort of unravelling mechanisms that Dr.
genome instability both in normal and cancerous cells.
So in the Haber lab, we use Saccharomyces cervisiae or budding
yeast as a model Organism to study DNA double strand breaks.
And so this image right here on the left is a nice microscopic image showing sort
of this phenotype of this Organism, which is a budding phenotype over here.
So it's characteristically called as such.
And so this is a unicellular fungus and it reproduces asymmetrically.
So you can see that this budding over here is a daughter cell coming out of a
mother cell.
And on the right here, it's illustrated very nicely.
And I really like this image put in in parallel because you can sort of see this
nice cell over here is probably in its G2M phase of the cell cycle.
So this is just the cell cycle of a haploid yeast cell.
Another feature of budding yeast is the ability to convert some cells in a
population from one mating type to another,
and this is shared with other yeast cells as well.
So budding yeast have two mating types, mat A and mat alpha,
and you can see it right over here.
This is a matte alpha cell and a matte A cell and they can propagate vegetatively
as such.
And mating type is determined by two different alleles at the mating type or
matte locus.
Additionally, budding yeast can also propagate
vegetatively as mat A and alpha diploids, and this can occur when mating of the
opposite mating types occur.
So a mat A cell mating with a mat alpha cell can form diploids.
And this is really evolutionarily advantageous because it provides features
that are not available to haploids.
Most importantly, these cells can undergo meiosis and
further spore formulation, which brings a grand genetic diversity
within a population.
And in the lab, this is really nice because it allows us
to do genetics really easily.
And so I'm just going to quickly go through this life cycle illustrated over
here.
So here we can see a mat, A cell dividing and giving rise to a
daughter cell right over here.
And what's really nice and what I find really interesting is that in nature,
the daughter cell has this sort of green dot over here,
which is indicative of its nucleus.
And so it contains something different from its mother cell.
And what's different about it is that this daughter cell cannot undergo mating
type switching, but its mother cell can.
And so that's what we can see over here.
The cells that it gives rise to are still mat a as it started off with,
but this mother cell has switched its mating type.
So it is now a matte alpha cell.
And I just told you a couple seconds ago that a matte A cell and a matte alpha
cell can mate with each other and form diploids.
And so in nature, this is exactly how a budding yeast can
sort of adapt to its environment really nicely.
And so here we can see that diploids have been formed and have can undergo meiosis
and sporulation and this whole process can continue to happen.
Now I'm going to focus now primarily on this sort of aspect over here of mating
type switching.
So you can see over here it says HO induced mat switching in late G1.
So the question is how exactly does this happen?
So remarkably, mating type switching is initiated by
adna double strand break and it's initiated at the mat chromosome.
And so many type switching is a really highly,
highly choreographed process that has taught us a lot about gene regulation,
chromosome structure and importantly homologous recombination.
And so some of you are probably wondering how exactly does a double strand break
lead to a mat a cell switching over to a mat alpha cell or vice versa?
So that's the question I'm going to answer in in the next couple of minutes.
So here is the arrangement of the map chromosome.
So this is this circle over here is indicative of its centromere.
It's very small, but I'm going to highlight just a few
things.
So first, this MAT A locus over here is
transcriptionally active.
That means that the sequences that are encoded here can be expressed and produce
proteins that give a MAT a cell mat A like characteristics.
And vice versa.
If we look at a MAT alpha cell, you can see that this area right over
here is transcriptionally active.
So this is a MAT alpha cell giving rise to MAT alpha like phenotypes.
And so importantly the the MAT locus contains 4 regions which we call WXY and
Z.
But there are other regions within this chromosome that you can see here called
HML and HMR.
And HMR contains the exact same sequences as MAT A cell or homologous sequences.
But the important caveat here is that HMR and HML are heterochromatic or
transcriptionally silenced.
So these sequences that although are homologous are never expressed.
And so additionally, HML also contains those same sequences,
but it is like a matte alpha cell.
And so I find this system to be very beautiful because it, I,
I find it remarkable how this can even sort of occur.
So a mate type switching is initiated by adna double strand break.
And importantly, it's initiated by this endonuclease
called HO endonuclease.
That HO stands for homothalism, which is just the term for how yeast can
sort of convert cells in a population from one mating type to another.
So that's what the HO stands for.
And so it recognizes a specific sequence founded between this Y&
Z junction right over here.
And when that break occurs, it is repaired through homologous
recombination using those heterochromatic donor templates that I mentioned.
And so depending on the mating type, it will use different donor templates.
So here a matte A cell will switch on over to a matte alpha cell using this HML
donor template over here.
Now, if it was a matte alpha cell switching to
a matte A cell, it would use this HMR heterochromatic
donor template to repair its break.
And so this is simply just a switching event, a replacement of sequences.
And here I put it up in a much more simplistic view.
And what's really remarkable to me is that the difference between a matte A
cell and a matte alpha cell, those different sequences are just 700
base pairs in length.
So in its entire genome, it contains the exact same sequences
except for those 700 base pairs of sequences.
And so like I said, a MAT A cell can switch to a MAT alpha
cell or vice versa.
And so I want to take a closer look at these heterochromatic donor templates
over here.
So like I said, they're homologous to the MAT chromosome
over here.
The only difference between these two is that HMR doesn't contain that W region
over here, but HML does.
And what's important is that these little lines just sort of represent that this is
a transcriptionally silent area.
So in the lab, we've sort of leveraged this system and
we can conditionally induce adna double strand break using a GAL HO system.
And So what that means is that we have this HO gene and this encodes for that HO
endonuclease and we have attached a galactose inducible promoter to this HO
gene so we can insert this into the yeast cell.
And what this means is that when galactose is not present in the media of
a yeast cell, that HO gene is turned off.
So that HO endonuclease is never expressed.
However, if we were to add galactose to that media,
that means that that HO gene can be turned on and therefore that HO
endonuclease is produced and can recognize that sequence and create adna
double strand break.
And what's cool about this is that for that specific sequence,
we can sort of move that around anywhere that we want.
So we can do a lot of cool genetic tools.
All right, so now let's get into the good stuff,
which is the steps of meaning time switching.
So how exactly is that double strand break repaired?
So I already mentioned that homologous recombination is the mechanism that is
used.
There are other mechanisms.
There is something called non homologous and joining,
which is when those ends of the break are simply just ligated back together.
However, in this process, because there are homologous sequences
present, it's not the preferred method since it is
rather mutagenic and error prone and homologous recombination on the other
hand is not OK.
So the first step is that HO endonuclease recognizing that sequence found in
between that Y&Z junction like I said.
And so it'll create a double strand break and I'm going to switch on over to
illustrating things in this way since it it'll highlight a few features of of
mating type switching.
So these two lines over here represent the two DNA strands.
OK.
So that second step is something that we call and resection,
which is simply when there are exonucleases that will cleave off the DNA
in a 5 prime to three prime manner.
So therefore it leaves behind single stranded DNA that is in in the three
prime direction.
And the third step is really, really important for mating type
switching, but moreover important for homologous
recombination in general, which is that this RAD 51 protein will
come and bind to that single stranded DNA.
So rat 51 is not just the protein that is found in budding yeast.
It's also found in humans and it functions in the same way where it's
mediates homologous recombination.
It's also found in bacteria and E coli.
It's called Wreck A, and it also functions in that same way.
So I just wanted to highlight sort of the conservation and how important this
protein is.
And in studies where you delete RAD 51, you don't get repaired through homologous
recombination.
And unfortunately if there is no homologous donor template present,
it will use that non homologous and joining mechanism,
but it is rather mutagenic.
So a lot of cells don't survive in certain contexts.
So the 4th step is that homology search, which is mediated by that RAD 51 protein.
So the way that I like to think about this is in its entire genome.
Try to imagine like a needle in a haystack,
that's what that RAD 51 protein is doing.
And somehow it finds it.
And so when it finds that donor template, a structure called a displacement loop or
AD loop is formed.
And So what that means is that it you, if you think about DNA replication and
you have those two DNA strands, it sort of opens it up.
And so this donor template is disrupted for a time being and it's used for repair.
And so the fifth step is new DNA synthesis that will occur in a 5 prime to
three prime manner where you can see that these little dots over here are
indicative of of new sequences being copied on over.
And so this intermediate right here is that important step that I mentioned of
sort of switching or replacement of sequences where you can see we're
switching from red sequences, a mat A cell over to these blue sequences
which contain mat alpha sequences.
And so what's important here is that you can see that this red sequence right over
here is considered to be non homologous.
So it needs to be removed.
So there are exonucleases that will come and remove that.
And then the 6th step is that displacement loop getting resolved and
that donor template will remain completely unchanged.
So despite it getting disrupted, those complementary strands getting
removed from each other, nothing will happen to that donor
template after the correct sequences are copied on over.
And then second strand synthesis happens where you can see I sort of should have
made it a little bit longer, but these are homologous sequences.
So they will pair up with each other and then DNA synthesis will continue on over
in a 5 prime to three prime manner.
So then in the end, we're left with newly synthesized DNA on
the recipient strand and that donor template remains unchanged.
So you can see now that we have switched on over from a matte A cell to a matte
alpha cell all through that just switching or replacement of sequences.
And what I hope that you can all appreciate is just how choreographed this
process is.
And I, I failed to mention several like steps
only for the lack of time, to be honest with you.
But if you have any questions, please let me know.
But this has been decades of research both by Jim Haber and members in his lab,
as well as other labs all across the world.
And so this is a really nice experiment that was done back in 1988 by Jim Haber
and two other amazing scientists that are up here.
And So what they did was they were able to physically monitor that mating type
switching event.
So they were able to see the replacement of those sequences.
And what they did was a Southern blot analysis or adna analysis.
And all that means is that you can sort of see certain DNA fragments of interest.
And So what this does is it makes use of restriction enzymes that are found all
throughout the genome.
And so that's what this S stands for.
It's a specific restriction enzyme that will cleave off this segment of DNA.
And then what you can do is you can use probes,
which are labeled DNA fragments that can identify specific sequences in a sample.
And so here what we're looking at is time.
So you can 012 and four hours.
And so here we're looking at a MAT A cell at 0 and what they have done is they have
added that galactose, so they have turned on that HO gene.
So therefore that HO endonuclease can come and cleave right over here,
this galactose induced HO endonuclease.
And what you can see here is that there is cut DNA and then after some time there
is new DNA found and that is a MAT alpha DNA.
And you could start to see it a little bit at two hours,
but you could really distinctly see it after four hours.
OK, so I'm going to switch gears a little bit
now and talk a little bit about chromatin.
So if we were to decondense a chromosome, we will find that there is chromatin,
which is a complex of DNA and proteins that serves as the functional form of of
genetic material.
And this allows for a way to for a cell to compact all of its DNA in an efficient
way.
But importantly, it allows for gene regulation.
And so the basic unit of chromatin consists of DNA wrapped around these
proteins called histones.
And it's specifically wrapped around a histone octomer.
And it's what's needed for about 150 base pairs or so of DNA to wrap around.
And so you can see that the histone octomer makes up it.
It makes up two copies of H2BH2AH3 and H4.
And what's important is it can form this structure called a nucleosome,
which is just the basic repeating unit of chromatin.
And so in eukaryotes, there are two chromatin structures that
exist called eukromatin and heterochromatin.
So eukromatin is characterized as loosely packed transcriptionally active regions
in the genome.
So the DNA is accessible and that gene is active.
In contrast, heterochromatin is characterized as
densely packed, transcriptionally inactive regions in the
genome.
So therefore that DNA is inaccessible and that gene is active.
In budding yeast, there are thought to be 3 major regions
of transcriptionally silent or heterochromatic areas.
I'm not going to talk about these two today because what's really of interest
to me is that silent mating type loci.
And so let's look a little bit closer at what's really of interest to me,
which is that HML heterochromatic donor over here.
And so I already showed you this genetic arrangement earlier,
but I'm just going to highlight a few different things that I didn't previously.
So there are three things that are really important for this HML locus to become
transcriptionally inactive.
The first is these E and I silencers, and so these encode sequences that DNA
binding proteins can recognize and bind to.
I honestly forgot what E stands for, but I stands for important for silencing
and so this was when it was discovered.
However, you can see that it contains the same
genetic arrangement as a matte alpha cell.
So it's WXY and Z.
And so the second thing that's important for silencing at HML are those DNA
binding proteins that can bind to those E and I silencers.
And then the third thing are well positioned nucleosomes across this HML
locus.
And so this is an experiment that was done way back in 1998.
And So what they were able to do is map the histones across HML.
And so you can see here that there are some well positioned nucleosomes.
However, there's also some less defined
nucleosomes around here.
But what's important here is that across the the regions that are really important
for silencing, specifically sort of where this HO endo
nucleus can recognize, there are well positioned histones around
that area.
So here we're looking at a zoomed in picture of this E silencer.
So from here all the way to here.
And what I'm going to talk about now is sort of how that heterochromatin
structure is established.
So I already told you that that E silencer encodes 4 sequences where DNA
binding proteins can come and bind.
So that's what these 3 grey circles indicate.
And these DNA binding proteins can recruit another set of proteins that are
very, very important for silencing and they're
called silent information regulatory proteins or SER proteins.
And so there are four different SER proteins.
And so one of these DNA binding proteins will recruit SER one,
which will then recruit SER 4, SER 3 and then importantly SER 2.
Now SER 2 is a histone deacetylase.
And So what that means is that it can catalyze a reaction where it can remove
an acetylation mark.
And so specifically on these well positioned histones across HML,
there are many post translational modifications, but what's really,
really important for silencing is this H3KH4K16 acetylation.
And for those of you that don't speak this language,
what this means is that this H4 protein on its 16th lysine residue,
there's an acetylation modification that was placed there.
And so Sir Two, what it will do, it was it will recognize this specific
modification and it will deacetylate it.
And what this allows for is Sir 3 to come and bind,
which will then recruit Sir 2 and then continue on in a subsequential manner.
We'll recruit Sir 3 and so forth.
And So what you can start to see is sort of how this heterochromatic structure is
is established.
And so this is a very stable structure.
And so there there's a lab over at UC Berkeley called Jasper Rhines Lab where
they have developed this genetic assay to measure loss of silencing at HML.
So they wanted to see well how stable is that heterochromatic structure at HML.
And so they did something very clever where they made use of genes that are
found here at Y.
And what I failed to mention earlier about those genes that are encoded within
MAT A or MAT alpha, which are also found again at HML and HMR,
is that there are two of them called Y A2 and Y A1.
And So what they did was they simply replaced that Y A2 gene with the Cree
recombinase gene.
And so this is again a heterochromatic structure.
And So what this allows for us to do is basically see if that create gene is ever
expressed, it will mediate the recombination of
these two locks P sites.
So the Cree recombinase protein is an enzyme that can recognize these locks P
sites.
And this is a really nice genetic tool because what it does is you can use these
locks P sites and sort of Orient them in different ways.
This has been studies that were done many decades ago,
and essentially what they have done here is they've oriented them in a way where
if Cree is ever expressed, it will mediate the recombination of
these two locks piece sites and therefore excise anything that is found in between
these two locks piece sites.
So what you can see here is that you have a cell that has all these genes here.
However, if there's a loss of silencing, you will lose these two genes.
And So what this does is that if a cell has that heterochromatic structure
maintained, meaning it is stable, cells will be red.
So if I look under a fluorescent microscope, I will see that they are red,
and that is because they have this RFP gene and that encodes for something
called a red fluorescent protein.
Additionally, they are resistant to this drug called G4
O 18.
And this is just really nice in the lab where you can just do a bunch of
different things with different media.
And So what this does is it allows for us to visually see a cell go from red to
green.
And so for those of you that don't know sort of how yeast lab works,
this is also true for many other organisms.
But what we can do is we can take an Agar plate,
which is essentially a sort of gel nutritional media.
And what I can do is I can take a single cell and I can plate it on that Agar
plate and I can wait maybe like two or three days.
And what I will be able to see is visually a colony.
And what that is, is a bunch of different cells that have
grown up for me to visually see it.
And so here is a colony of a single cell where you can see it.
It is, it is red and it has this little sort of
stripe over here that is green.
And so that means that like some cells that have sort of formed to make this
colony have lost silencing.
And what was novel about sort of using this genetic tool is that about one to 2%
of cells lose silencing during normal replication.
So what that means is that some cells that have grown up into that colony seem
to have lost silencing.
And it gave this phenotype over here of half green and half red,
because what this means is that either the first or second cell that gave rise
to that colony lost silencing.
So this brings me to sort of my research project and what I'm really interested in,
which is to sort of answer this question of, well,
how is the stability of heterochromatin maintenance affected by homologous
recombination?
And broadly, how is that regulated in the cell?
And So what I can do is I can use that crash assay and modify it to do several
different things.
And So what I'm really interested in is this intermediate right over here where I
told you that that displacement loop can be formed.
I'm really interested to understand how exactly does that donor template open up
and allow for that to happen.
Since it is a heterochromatic structure and it is stable,
how exactly does that change to allow for repair to happen and why does it do that?
Why does it do that in in this context but not in other contexts?
And So what we have done is we have constructed this pseudo map,
a fragment over here and inserted it on a different chromosome.
So you can see I've inserted it into chromosome 8.
And So what this contains is a truncated Cree gene right over here.
And So what this allows for is just the reporter system to to work because if you
can imagine that Cree gene is always expressed,
that means that cells will always be green.
So it doesn't give us a measurement, a readout of loss of silencing.
Additionally, it contains that Y A1 gene, this Z,
and then a little bit of that HML and in total it makes up 2.
8 kilobases of homology between pseudomat and HML.
And So what I can do is I can use that Galicho system to conditionally induce
adna double strand break at the pseudomat locus and then I can ask a question, well,
what happens to silencing after I induce a double strand break?
So what I find is that about 50% of cells experience a loss of silencing event at
HML Cree.
And specifically this is about after four hours.
So here we're looking at 0 where I didn't add galactose and then this is half an
hour all the way to 4 hours after adding that galactose and,
and conditionally turning on that HO endonuclease.
And so this brings up a lot of questions, which is, well, why is it 50% of the time?
Why is it not more?
Why is it not less?
So there's a lot of intricacies to, to work out since it's not simply adna
repair event that I'm looking at.
I'm also looking at a transcriptional event, right?
I'm looking at a transcriptional event from that HML locus itself.
And so I'm going to tell you two things that I am currently working on to work
out those intricacies.
And so the first thing that I'm really interested in is sort of assessing, well,
how much of an effect does the length of homology have on that loss of silencing
outcome.
So what I want to do is I want to decrease that homology.
And so I want to delete this truncated Cree over here and see what effect does
that have to the loss of silencing.
Since I told you earlier, and I'm going to go back to sort of this
step right over here, is that the really important part of
double strand brake repair is, is sort of this copping over of those
sequences.
And what's really cool about mating type switching is it sort of knows when to
stop almost.
And so I want to assess that in my own system and see what will happen if I
delete that cregene.
Well, how far does it go?
Will I get less loss of silencing outcomes?
Does it not make a difference?
So this is just one intricacy that I'm currently working on.
The second thing is I want to design a system that sort of mimics normal mating
type switching.
So here in this system, we're not looking at a replacement of
sequences.
It's just copying over the same sequences that have been broken, right?
And So what I would like to do is I would like to replace that A1 sequence with
that A1 sequence and assess what happens to to loss of silencing,
since this will sort of mimic normal mating type switching.
And therefore we can sort of learn a little bit more about mating type
switching and add on to decades of research that was done by both Jim Haber
and many other labs in the world.
And so with that, I would like to just quickly summarise
everything that I went over.
So mating type switching and budding yeast depends on a a double strand break,
which is repaired through homologous recombination using those heterochromatic
donor templates.
There are many steps involved in mating type switching.
The dynamics of heterochromatin establishment and maintenance can be
measured through that crash assay.
After inducing a double strand break, about 50% of cells experience a loss of
silencing.
And then lastly, my research questions I hope will provide
insight into how chromatin structure effects recombination and gene expression.
And so now it's time for some acknowledgments.
So I would like to 1st thank the two labs that I am in,
the Haber and Ragunathan labs.
Jim Haber and Kaushik Gurgunathan are amazing mentors and I'm so lucky to have
2P is honestly.
And then I would also like to thank the community that have build at Brandeis,
especially the friends that I have made who are actually here,
right here in the front seat.
So here is Shell and I in the lab.
And then lastly, I would like to thank my family.
Only one is here today, my mom, but my dad is on FaceTime.
My little brother is in high school right now.
But yeah, with that, I would take any questions after you are.
Yeah, and thank you all for listening as well
as someone who has worked with the Haver lab is someone who goes to Haver, Jim.
He really is tough on students whom he cares about and he cares a lot, right.
So it is a very fast-paced environment and you learn a lot,
You do a lot in a very short amount of time.
And you started your PhD straight out of here.
So you had your experience here, so can you advise students who wanted to
pursue a PhD in high school that is highly competitive and research focused
as fertilized university, what is some advice you have for people
that want to pursue a path like yours?
Yeah.
So I would say what really sort of helped me the most is seeking out sort of
different kind of opportunities.
So the first of course was joining your lab and getting exposed to to to research
in a small way through your lab.
Additionally, I would also say sort of finding
community within your professors.
So as as you know, all of my professors that are here that I
made a community out of sort of helped me a lot.
I would also say that it's, it may sound scary,
however I think that like there's proof right in front of me right now,
which is you will make a community at whatever sort of school you decide to go
to.
This is proof right over here.
They came all the way from Waltham to to come and watch me talk here.
And additionally, it's, it's not, it's not just you doing everything on
your own.
It may sound sort of like, oh, well, you're the one doing all this work with
you and your Pi, but you're also in a lab.
And there's also labs next to you, above you, below you.
And so you're, you're going to get support through many
different avenues.
But yeah, I think, I think that's, that's the advice that that I would give.
Yeah, yes.
Truly wonderful call.
Thank you.
I'm curious about the idea of the switching is actually an invasion and
then copying a minute withdrawals, right, correct.
There's lots of questions about how far in that gather you're trying to figure
that out.
But how does that compare?
What happens differently when you have monologous recombination 'cause you're
not doing homologous recombination, copying is not changing anything,
but in homologous recombination, things are exchanged.
So what?
How does this remain?
Yes, yeah, yeah, yeah, yeah.
So right there, there are many different sort of kinds of
homologous recombination.
Yes.
So I think the kind that you're thinking about sort of happens when there's a
crossover event, right?
When there's like there's actual like exchange of DNA sequences, right.
So this kind of homologous or combination doesn't have that sort of exchange
because it is a different kind.
And those do exist.
Yeah.
So there, there are definitely other contexts in
which that does occur and which it's not in meeting type switching obviously since
nothing happens to that donor template.
But there are many other different contexts in which we have learned that
sort of crossover event.
But in this specific situation that doesn't happen remarkably.
It might happen maybe in a small percentage of cells,
but but not enough where we can sort of detect it through various tools that we
use to detect it, You know, so we can sort of screen different way
different outcomes that happen, right.
So this specifically is is called sort of a gene conversion event.
And we can do a bunch of different things to sort of analyze that.
One simple way is just by doing APCR, honestly.
And so if we were to do that, we wouldn't be able to detect that small
percentage of some cells that maybe have crossed over.
But I it's really unlikely just based on like past experiments that have been done.
But yeah, that's, that doesn't occur in this context.
Right over here.
Yeah.
Thank you for a great talk.
And forgive me, I'm a cell biologist.
I'm at the organization level, but I am, but you're talking so good that I'll be
able to follow it.
And however my question I hope makes sense.
I am just curious as to, you know, it sounds like some of the yeast can mate
switch and some cannot.
And so given what you shared that I was wondering if the ones that cannot,
are they the ones that have the served proteins finding to like silence that
area that can't be expressed?
Yeah.
So, so some cells that that can't switch.
So I think the the ones that that can't switch probably have some kind of yeah,
some kind of defect in, in some kind of small aspect in that
system itself.
Whether it be some sort of defect by like a mutation in in some important protein
for that heterochromatic donor template itself.
Or it could be something within that expressed locus itself where perhaps
maybe that recognition site is removed in a way or like, you know,
that that HO endonuclease can't cleave there.
So therefore mating type switching cannot happen.
It can't be initiated in the 1st place.
So, yeah, I think those are maybe two different
areas in that whole process that perhaps causes some kind of effect in some cells
not being able to switch.
I'm just curious how much I OK, thank you.
I'm just curious how much is known about how RAT 51 actually helps in the scanning
to look for Mollus sequences?
How much we know?
Yeah, I'm just like, what's the mechanism?
Yeah, yeah, yeah.
That's a.
That's a loaded question and it's.
It's a great question and it is a question that researchers are still
trying to answer to this day.
It is, it's, it's remarkable, right?
How can, how exactly can it find a needle in a
haystack?
You know, the, the things that we do know have mostly
come from deleting RAD 51 and then seeing what happens in,
in several different kind of repair mechanisms, right?
So I just told you about 1:00.
But that is also important in many of other different ones.
And when you delete that protein, you just, you don't get repair.
So therefore cells eventually die.
And so that's how we know that this protein is is very, very important in,
in repairing a break at least.
But yeah, I like we, we still don't know so much.
It is still still being researched today.
I, I asked my Pi that sort of like the first
time that I was learning all this and joined his lab and he was like,
I don't have an answer for you.
Like this is still things that we are trying to figure out.
Can I just ask a follow up question?
Yeah, Are there, are there they're like mutants that are
not null mutants of RAD 51 that like impair the function that don't totally
get rid of it.
That might give some clue.
Yeah, absolutely.
Yeah, there are definitely those kind of
mutants that have been that have been screened.
I I can't remember off the top of my head sort of the different phenotypes that
like they give you, but but they're they are out there.
Yes, I know that for certain.
Yeah, yeah.