WEBVTT

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From Salem State,
the BS Biology and an option in

d2f50aad-9fbf-41b0-8bd8-b6219517baa5-1
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Biomedical Science at the Chemistry minor
in May of 2023.

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While an undergraduate,
Pamela's biological curiosity was piqued

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by numerous courses here.

91ecb803-5f4e-4070-9abe-b317f3f2670d-0
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What further cemented her desire to
become a research scientist?

5de6700e-990c-4b6c-aa8b-2fa1a2ee94de-0
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For two summer research experiences,
I think it was two of them.

77786af0-89b4-4af9-b856-a64f1698889c-0
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At the Ragon Institute,
working with Doctor Schiff Pillai,

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I chatted with Pamela's Mama.

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Money would go.

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Pamela, like me, likes tennis time.

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I'm going to the gym,
but she's very busy doing her PhD at home.

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But you aren't playing as much tennis,
I hear.

81e9dd51-cc32-4172-aec3-fc499c236732-0
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No, unfortunately not.

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Pamela is our PhD candidate at Brandeis
University and today she'll tell us about

a6d62edd-3068-4a5f-9d89-fd7b7e6d5d15-1
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her research and her talk entitled The
Intersection of Homologous Recombination

a6d62edd-3068-4a5f-9d89-fd7b7e6d5d15-2
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and Heterochromatin Maintenance in the
Sacrifice.

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Sarah Vizier.

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So welcome back, Pamela.

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Thank you so much for that lovely
introduction.

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So yeah, my name is Pam.

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I am in the Haber and Ragunathan labs at
Brandeis University,

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and I am a second year PhD student in the
Molecular and Cell Biology program.

f732370d-1c2d-4f82-8c23-9c31a215c71d-0
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So this festival in in Salem State in
general holds a very special place in my

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heart.

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I made a great community here and I am
still in touch and love all of the

56be4cfe-461c-4612-b415-9053453b3270-1
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professors that taught me a lot of things
and sort of paved the way for Graduate

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School for me.

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So I'm very excited to tell you about my
research project and how I'm beginning it.

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So I am in both the Haber and Ragunathan
labs,

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but I'm going to be talking to you all
about research mostly in the Haber lab.

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So to begin, when we were born,
we inherited 23 pairs of chromosomes from

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our parents.

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And this was arranged in a very specific
way.

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And despite through going through
countless cell divisions,

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that arrangement has remained largely
stable, ensuring proper development and,

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and function.

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And so over here,
let me put the laser over here,

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we're looking at a karyotype or a visual
representation of chromosomes where we

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can see the number, size and structure.

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And here we're looking at a normal
individual.

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And so this ability of of a cell to
maintain its integrity of its genomic

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material is,
is really important because it prevents

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mutations and chromosomal rearrangements.

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Now the exception to this, of course,
is what happens in tumor cells.

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So that stability is disrupted and that
leads to a lot of genomic instability,

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while cells can develop mutations and
chromosomal abnormalities.

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So here we're looking at a karyotype of a,
of a cancer cell where you can see that

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that three distinctions that I told you
are very, very different.

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You can see that there are a lot more
chromosomes, they look very different.

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And so a serious threat to genome
stability is adna double strand break.

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And so in the Haber lab,
we are interested in studying DNA double

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strand breaks.

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And so if we were to zoom into the
nucleus of a cell,

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we would find this chromosome.

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And if we were to decondense it to expose
the naked DNA,

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we would find this DNA double helical
structure over here.

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And so a double strand break is just a
kind of DNA damage in which both of those

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DNA helixes can break.

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And when that breaks,
that kind of damage can lead to a lot of

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serious threats.

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And if that isn't correctly repaired,
it can lead to mutations and chromosomal

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rearrangements.

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So understanding how double strand breaks
are generated and repaired is really

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important to sort of unravelling
mechanisms that Dr.

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genome instability both in normal and
cancerous cells.

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So in the Haber lab,
we use Saccharomyces cervisiae or budding

20a25215-2568-44fe-9f90-28c176a40885-1
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yeast as a model Organism to study DNA
double strand breaks.

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And so this image right here on the left
is a nice microscopic image showing sort

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of this phenotype of this Organism,
which is a budding phenotype over here.

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So it's characteristically called as such.

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And so this is a unicellular fungus and
it reproduces asymmetrically.

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So you can see that this budding over
here is a daughter cell coming out of a

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mother cell.

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And on the right here,
it's illustrated very nicely.

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And I really like this image put in in
parallel because you can sort of see this

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nice cell over here is probably in its
G2M phase of the cell cycle.

ab411cd6-ac61-4fcf-83d8-670b251f2257-0
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So this is just the cell cycle of a
haploid yeast cell.

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Another feature of budding yeast is the
ability to convert some cells in a

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population from one mating type to
another,

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and this is shared with other yeast cells
as well.

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So budding yeast have two mating types,
mat A and mat alpha,

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and you can see it right over here.

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This is a matte alpha cell and a matte A
cell and they can propagate vegetatively

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as such.

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And mating type is determined by two
different alleles at the mating type or

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matte locus.

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Additionally,
budding yeast can also propagate

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vegetatively as mat A and alpha diploids,
and this can occur when mating of the

4a5a4013-3720-46a5-828b-98f884127010-2
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opposite mating types occur.

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So a mat A cell mating with a mat alpha
cell can form diploids.

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And this is really evolutionarily
advantageous because it provides features

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that are not available to haploids.

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Most importantly,
these cells can undergo meiosis and

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further spore formulation,
which brings a grand genetic diversity

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within a population.

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And in the lab,
this is really nice because it allows us

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to do genetics really easily.

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And so I'm just going to quickly go
through this life cycle illustrated over

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here.

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So here we can see a mat,
A cell dividing and giving rise to a

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daughter cell right over here.

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And what's really nice and what I find
really interesting is that in nature,

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the daughter cell has this sort of green
dot over here,

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which is indicative of its nucleus.

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And so it contains something different
from its mother cell.

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And what's different about it is that
this daughter cell cannot undergo mating

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type switching, but its mother cell can.

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And so that's what we can see over here.

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The cells that it gives rise to are still
mat a as it started off with,

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but this mother cell has switched its
mating type.

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So it is now a matte alpha cell.

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And I just told you a couple seconds ago
that a matte A cell and a matte alpha

f9971d84-4d12-40f4-8d48-fe82302a1919-1
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cell can mate with each other and form
diploids.

f58d1832-7610-4e0e-b6b6-06c7de7b464c-0
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And so in nature,
this is exactly how a budding yeast can

f58d1832-7610-4e0e-b6b6-06c7de7b464c-1
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sort of adapt to its environment really
nicely.

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And so here we can see that diploids have
been formed and have can undergo meiosis

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and sporulation and this whole process
can continue to happen.

289a9f05-c979-4092-9f0c-cc18fe0d1b92-0
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Now I'm going to focus now primarily on
this sort of aspect over here of mating

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type switching.

fd9b04c4-4b3e-431d-bdd0-6cb0aa2e61e1-0
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So you can see over here it says HO
induced mat switching in late G1.

7ddc8b0f-bd57-4efa-8a3a-a2a5269370c8-0
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So the question is how exactly does this
happen?

a72d3d15-fcd8-4b54-89f9-1a8bcd4264f1-0
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So remarkably,
mating type switching is initiated by

a72d3d15-fcd8-4b54-89f9-1a8bcd4264f1-1
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adna double strand break and it's
initiated at the mat chromosome.

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And so many type switching is a really
highly,

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highly choreographed process that has
taught us a lot about gene regulation,

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chromosome structure and importantly
homologous recombination.

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And so some of you are probably wondering
how exactly does a double strand break

7e8f1c68-3404-456b-872d-0637f93846fd-1
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lead to a mat a cell switching over to a
mat alpha cell or vice versa?

b4307afa-5853-4274-a1a1-29791547eaa0-0
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So that's the question I'm going to
answer in in the next couple of minutes.

b2dbef1b-996e-4897-9730-b03f28432835-0
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So here is the arrangement of the map
chromosome.

aa7d1611-5083-4fbc-b935-391dd198d006-0
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So this is this circle over here is
indicative of its centromere.

1ba043c3-726b-4047-be2a-186450b035bc-0
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It's very small,
but I'm going to highlight just a few

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things.

f9a74e16-d42b-420b-8e9a-329ac6d5aa86-0
00:08:14.960 --> 00:08:18.061
So first,
this MAT A locus over here is

f9a74e16-d42b-420b-8e9a-329ac6d5aa86-1
00:08:18.061 --> 00:08:20.000
transcriptionally active.

029f8666-c77b-460e-8fa6-e3c5d2dbdfa8-0
00:08:20.200 --> 00:08:24.657
That means that the sequences that are
encoded here can be expressed and produce

029f8666-c77b-460e-8fa6-e3c5d2dbdfa8-1
00:08:24.657 --> 00:08:27.960
proteins that give a MAT a cell mat A
like characteristics.

3d0eaaac-3e51-4996-aa47-b71c41561c69-0
00:08:28.200 --> 00:08:28.920
And vice versa.

46400ea8-d91e-4870-b1e0-d9027fbf30b5-0
00:08:28.920 --> 00:08:32.562
If we look at a MAT alpha cell,
you can see that this area right over

46400ea8-d91e-4870-b1e0-d9027fbf30b5-1
00:08:32.562 --> 00:08:34.280
here is transcriptionally active.

07552df1-ec5f-4214-845b-b3387f4f9d1e-0
00:08:34.280 --> 00:08:38.200
So this is a MAT alpha cell giving rise
to MAT alpha like phenotypes.

1a9732f4-1b08-44b3-8f6c-359f0e687473-0
00:08:38.880 --> 00:08:45.471
And so importantly the the MAT locus
contains 4 regions which we call WXY and

1a9732f4-1b08-44b3-8f6c-359f0e687473-1
00:08:45.471 --> 00:08:45.640
Z.

ef601777-d672-4319-8258-6664282f3270-0
00:08:47.360 --> 00:08:51.916
But there are other regions within this
chromosome that you can see here called

ef601777-d672-4319-8258-6664282f3270-1
00:08:51.916 --> 00:08:52.600
HML and HMR.

c8527bbc-e081-42e2-bf6b-79213719f945-0
00:08:54.040 --> 00:09:00.120
And HMR contains the exact same sequences
as MAT A cell or homologous sequences.

54ce5326-fced-4b86-9011-fb73f8643d58-0
00:09:01.120 --> 00:09:05.792
But the important caveat here is that HMR
and HML are heterochromatic or

54ce5326-fced-4b86-9011-fb73f8643d58-1
00:09:05.792 --> 00:09:07.520
transcriptionally silenced.

356632c9-3c55-4b17-b57b-e7300d8febc3-0
00:09:07.520 --> 00:09:11.720
So these sequences that although are
homologous are never expressed.

1ded8b27-3ab9-4ab4-88ba-e8521210071b-0
00:09:12.520 --> 00:09:16.218
And so additionally,
HML also contains those same sequences,

1ded8b27-3ab9-4ab4-88ba-e8521210071b-1
00:09:16.218 --> 00:09:18.280
but it is like a matte alpha cell.

a7e92d7b-e641-45ba-a2b9-6b0c3856b4e4-0
00:09:18.960 --> 00:09:22.594
And so I find this system to be very
beautiful because it, I,

a7e92d7b-e641-45ba-a2b9-6b0c3856b4e4-1
00:09:22.594 --> 00:09:25.760
I find it remarkable how this can even
sort of occur.

c4c00c64-5460-492b-9426-7fcd6b666d1d-0
00:09:27.040 --> 00:09:30.880
So a mate type switching is initiated by
adna double strand break.

9df53d04-70a6-4ef4-a2dd-1a8f18c5f837-0
00:09:31.040 --> 00:09:33.941
And importantly,
it's initiated by this endonuclease

9df53d04-70a6-4ef4-a2dd-1a8f18c5f837-1
00:09:33.941 --> 00:09:35.200
called HO endonuclease.

9ae7ea45-dd66-4a40-9abf-b8829c2bd81d-0
00:09:35.760 --> 00:09:40.403
That HO stands for homothalism,
which is just the term for how yeast can

9ae7ea45-dd66-4a40-9abf-b8829c2bd81d-1
00:09:40.403 --> 00:09:44.920
sort of convert cells in a population
from one mating type to another.

7ffee868-ac77-42a4-b702-31e99720f778-0
00:09:45.040 --> 00:09:47.040
So that's what the HO stands for.

de05d487-fc9d-4d7c-81f1-aa26f6341beb-0
00:09:48.360 --> 00:09:52.097
And so it recognizes a specific sequence
founded between this Y&amp;

de05d487-fc9d-4d7c-81f1-aa26f6341beb-1
00:09:52.097 --> 00:09:53.560
Z junction right over here.

82ccb23a-df16-4339-ae80-ea336bd066a3-0
00:09:54.280 --> 00:09:57.119
And when that break occurs,
it is repaired through homologous

82ccb23a-df16-4339-ae80-ea336bd066a3-1
00:09:57.119 --> 00:10:00.600
recombination using those heterochromatic
donor templates that I mentioned.

e9b46ad7-06b2-4075-972d-560dde091f3f-0
00:10:01.640 --> 00:10:07.240
And so depending on the mating type,
it will use different donor templates.

21b566c4-8924-435d-b774-f270d45f5146-0
00:10:07.760 --> 00:10:12.849
So here a matte A cell will switch on
over to a matte alpha cell using this HML

21b566c4-8924-435d-b774-f270d45f5146-1
00:10:12.849 --> 00:10:14.440
donor template over here.

020a7018-3e02-4102-906c-f6e5b89e7f97-0
00:10:14.920 --> 00:10:17.477
Now,
if it was a matte alpha cell switching to

020a7018-3e02-4102-906c-f6e5b89e7f97-1
00:10:17.477 --> 00:10:20.415
a matte A cell,
it would use this HMR heterochromatic

020a7018-3e02-4102-906c-f6e5b89e7f97-2
00:10:20.415 --> 00:10:22.320
donor template to repair its break.

4cb4dd13-e5e7-4265-a954-4b23ed9f8457-0
00:10:22.680 --> 00:10:26.760
And so this is simply just a switching
event, a replacement of sequences.

590a906a-e75d-4077-99fd-5f4e668bdb2c-0
00:10:27.520 --> 00:10:31.360
And here I put it up in a much more
simplistic view.

a7a59d2b-cd12-43f7-9f6f-3c28939f6f29-0
00:10:31.960 --> 00:10:36.179
And what's really remarkable to me is
that the difference between a matte A

a7a59d2b-cd12-43f7-9f6f-3c28939f6f29-1
00:10:36.179 --> 00:10:39.954
cell and a matte alpha cell,
those different sequences are just 700

a7a59d2b-cd12-43f7-9f6f-3c28939f6f29-2
00:10:39.954 --> 00:10:41.120
base pairs in length.

8b7e834c-d208-46b7-822f-27154bb77154-0
00:10:41.840 --> 00:10:45.261
So in its entire genome,
it contains the exact same sequences

8b7e834c-d208-46b7-822f-27154bb77154-1
00:10:45.261 --> 00:10:47.800
except for those 700 base pairs of
sequences.

3fe38c77-d9f1-444a-b8a1-e9726edb7081-0
00:10:50.080 --> 00:10:54.073
And so like I said,
a MAT A cell can switch to a MAT alpha

3fe38c77-d9f1-444a-b8a1-e9726edb7081-1
00:10:54.073 --> 00:10:55.360
cell or vice versa.

ebb2a64e-752b-4bae-bc9f-ebc9e22199ec-0
00:10:56.800 --> 00:11:01.154
And so I want to take a closer look at
these heterochromatic donor templates

ebb2a64e-752b-4bae-bc9f-ebc9e22199ec-1
00:11:01.154 --> 00:11:01.720
over here.

4ab20534-1a01-43fa-af34-c6d41850a3a4-0
00:11:02.320 --> 00:11:05.552
So like I said,
they're homologous to the MAT chromosome

4ab20534-1a01-43fa-af34-c6d41850a3a4-1
00:11:05.552 --> 00:11:06.120
over here.

c17b9b17-f3f1-44d7-8f8c-92e1bce69cc2-0
00:11:07.360 --> 00:11:11.729
The only difference between these two is
that HMR doesn't contain that W region

c17b9b17-f3f1-44d7-8f8c-92e1bce69cc2-1
00:11:11.729 --> 00:11:13.040
over here, but HML does.

cc20914f-99e6-4116-8703-d79109615db1-0
00:11:13.840 --> 00:11:18.329
And what's important is that these little
lines just sort of represent that this is

cc20914f-99e6-4116-8703-d79109615db1-1
00:11:18.329 --> 00:11:20.040
a transcriptionally silent area.

5e7a4e43-ecd2-4d09-bd73-a45736b61805-0
00:11:22.840 --> 00:11:27.743
So in the lab,
we've sort of leveraged this system and

5e7a4e43-ecd2-4d09-bd73-a45736b61805-1
00:11:27.743 --> 00:11:34.520
we can conditionally induce adna double
strand break using a GAL HO system.

240c9300-6436-4b57-9484-340bd231c59e-0
00:11:35.000 --> 00:11:41.985
And So what that means is that we have
this HO gene and this encodes for that HO

240c9300-6436-4b57-9484-340bd231c59e-1
00:11:41.985 --> 00:11:48.540
endonuclease and we have attached a
galactose inducible promoter to this HO

240c9300-6436-4b57-9484-340bd231c59e-2
00:11:48.540 --> 00:11:52.680
gene so we can insert this into the yeast
cell.

8a8bb226-71eb-49fc-adcc-d1cc989b1369-0
00:11:53.800 --> 00:11:58.149
And what this means is that when
galactose is not present in the media of

8a8bb226-71eb-49fc-adcc-d1cc989b1369-1
00:11:58.149 --> 00:12:00.560
a yeast cell, that HO gene is turned off.

945a03dc-e6a9-4229-af0b-9bea18218b6c-0
00:12:00.560 --> 00:12:03.240
So that HO endonuclease is never
expressed.

ffd0b073-93f2-4012-8b9f-4d114374f744-0
00:12:03.680 --> 00:12:06.472
However,
if we were to add galactose to that media,

ffd0b073-93f2-4012-8b9f-4d114374f744-1
00:12:06.472 --> 00:12:10.125
that means that that HO gene can be
turned on and therefore that HO

ffd0b073-93f2-4012-8b9f-4d114374f744-2
00:12:10.125 --> 00:12:14.045
endonuclease is produced and can
recognize that sequence and create adna

ffd0b073-93f2-4012-8b9f-4d114374f744-3
00:12:14.045 --> 00:12:15.120
double strand break.

f1510623-0b10-46fe-a249-e10dede8800b-0
00:12:15.680 --> 00:12:18.370
And what's cool about this is that for
that specific sequence,

f1510623-0b10-46fe-a249-e10dede8800b-1
00:12:18.370 --> 00:12:20.720
we can sort of move that around anywhere
that we want.

c9f5fc81-1c95-4739-b446-09fbb6efb8b4-0
00:12:21.080 --> 00:12:23.280
So we can do a lot of cool genetic tools.

b6b86f6a-fdbe-40f4-9bbc-634273895d1c-0
00:12:25.320 --> 00:12:27.713
All right,
so now let's get into the good stuff,

b6b86f6a-fdbe-40f4-9bbc-634273895d1c-1
00:12:27.713 --> 00:12:29.960
which is the steps of meaning time
switching.

53f4a90e-6204-4d02-94ad-f17edd9a735a-0
00:12:30.800 --> 00:12:34.160
So how exactly is that double strand
break repaired?

007ef588-cd6c-49a6-bd4a-ed4466cc60a8-0
00:12:34.360 --> 00:12:38.269
So I already mentioned that homologous
recombination is the mechanism that is

007ef588-cd6c-49a6-bd4a-ed4466cc60a8-1
00:12:38.269 --> 00:12:38.520
used.

8e45923a-23ca-408b-9810-50c06164b41a-0
00:12:38.720 --> 00:12:40.120
There are other mechanisms.

c911e19c-0053-4cc3-b463-84590feaf038-0
00:12:40.560 --> 00:12:43.494
There is something called non homologous
and joining,

c911e19c-0053-4cc3-b463-84590feaf038-1
00:12:43.494 --> 00:12:47.680
which is when those ends of the break are
simply just ligated back together.

1edf9c0e-2528-4cab-b019-a304f071a223-0
00:12:48.040 --> 00:12:52.520
However, in this process,
because there are homologous sequences

1edf9c0e-2528-4cab-b019-a304f071a223-1
00:12:52.520 --> 00:12:56.036
present,
it's not the preferred method since it is

1edf9c0e-2528-4cab-b019-a304f071a223-2
00:12:56.036 --> 00:13:01.206
rather mutagenic and error prone and
homologous recombination on the other

1edf9c0e-2528-4cab-b019-a304f071a223-3
00:13:01.206 --> 00:13:02.240
hand is not OK.

b2502cdd-6a93-4694-8ee9-9bbe66eb60d8-0
00:13:02.400 --> 00:13:06.515
So the first step is that HO endonuclease
recognizing that sequence found in

b2502cdd-6a93-4694-8ee9-9bbe66eb60d8-1
00:13:06.515 --> 00:13:08.760
between that Y&amp;Z junction like I said.

b2a48ef0-effa-4adb-90a3-798b467d983a-0
00:13:08.960 --> 00:13:13.588
And so it'll create a double strand break
and I'm going to switch on over to

b2a48ef0-effa-4adb-90a3-798b467d983a-1
00:13:13.588 --> 00:13:18.277
illustrating things in this way since it
it'll highlight a few features of of

b2a48ef0-effa-4adb-90a3-798b467d983a-2
00:13:18.277 --> 00:13:19.600
mating type switching.

4b80d933-5a9f-4854-b77d-061c5488dbd1-0
00:13:19.600 --> 00:13:24.040
So these two lines over here represent
the two DNA strands.

1ba23efc-4c07-43f7-8ac8-2fe5f8e64284-0
00:13:25.560 --> 00:13:25.880
OK.

73875ad7-acba-4fad-9f58-d243b474217e-0
00:13:26.000 --> 00:13:29.912
So that second step is something that we
call and resection,

73875ad7-acba-4fad-9f58-d243b474217e-1
00:13:29.912 --> 00:13:34.595
which is simply when there are
exonucleases that will cleave off the DNA

73875ad7-acba-4fad-9f58-d243b474217e-2
00:13:34.595 --> 00:13:36.840
in a 5 prime to three prime manner.

a1e04561-e960-4557-ad0e-c4d01216454b-0
00:13:37.080 --> 00:13:41.651
So therefore it leaves behind single
stranded DNA that is in in the three

a1e04561-e960-4557-ad0e-c4d01216454b-1
00:13:41.651 --> 00:13:42.640
prime direction.

a144d7e4-fa6e-468e-a8b3-015fb0f0a2b3-0
00:13:44.720 --> 00:13:48.586
And the third step is really,
really important for mating type

a144d7e4-fa6e-468e-a8b3-015fb0f0a2b3-1
00:13:48.586 --> 00:13:51.593
switching,
but moreover important for homologous

a144d7e4-fa6e-468e-a8b3-015fb0f0a2b3-2
00:13:51.593 --> 00:13:55.582
recombination in general,
which is that this RAD 51 protein will

a144d7e4-fa6e-468e-a8b3-015fb0f0a2b3-3
00:13:55.582 --> 00:13:58.160
come and bind to that single stranded DNA.

bdba7466-d0fe-4454-b199-4f55147dfd5e-0
00:13:58.840 --> 00:14:02.360
So rat 51 is not just the protein that is
found in budding yeast.

71566b92-1637-4e49-8fdd-0f23d61c0ee0-0
00:14:02.480 --> 00:14:06.653
It's also found in humans and it
functions in the same way where it's

71566b92-1637-4e49-8fdd-0f23d61c0ee0-1
00:14:06.653 --> 00:14:08.680
mediates homologous recombination.

75cafb38-8bf9-406d-94ba-ca8b0dca9d54-0
00:14:09.120 --> 00:14:11.120
It's also found in bacteria and E coli.

ba96c318-18bf-4220-a200-1248b9a3fa2e-0
00:14:11.120 --> 00:14:13.760
It's called Wreck A,
and it also functions in that same way.

b34d2fe3-9459-450f-9eca-cf58ce71e11e-0
00:14:14.320 --> 00:14:18.000
So I just wanted to highlight sort of the
conservation and how important this

b34d2fe3-9459-450f-9eca-cf58ce71e11e-1
00:14:18.000 --> 00:14:18.520
protein is.

f93646f8-6358-4de9-ad2a-bd052ce170ce-0
00:14:19.760 --> 00:14:24.543
And in studies where you delete RAD 51,
you don't get repaired through homologous

f93646f8-6358-4de9-ad2a-bd052ce170ce-1
00:14:24.543 --> 00:14:25.360
recombination.

10386fba-c405-4eae-88f2-53138ce1fa2b-0
00:14:25.520 --> 00:14:29.581
And unfortunately if there is no
homologous donor template present,

10386fba-c405-4eae-88f2-53138ce1fa2b-1
00:14:29.581 --> 00:14:32.867
it will use that non homologous and
joining mechanism,

10386fba-c405-4eae-88f2-53138ce1fa2b-2
00:14:32.867 --> 00:14:34.480
but it is rather mutagenic.

cd3fc61c-58a1-4f6a-8a1b-7e97c0331f59-0
00:14:34.480 --> 00:14:37.240
So a lot of cells don't survive in
certain contexts.

de09800f-fbe2-4ea3-ac9b-c7e622ed5dc4-0
00:14:39.640 --> 00:14:44.600
So the 4th step is that homology search,
which is mediated by that RAD 51 protein.

406d8730-4fb9-4335-8f0b-97d761001574-0
00:14:44.600 --> 00:14:48.880
So the way that I like to think about
this is in its entire genome.

758d3751-6271-41ca-9a40-cda96ff08827-0
00:14:48.880 --> 00:14:51.012
Try to imagine like a needle in a
haystack,

758d3751-6271-41ca-9a40-cda96ff08827-1
00:14:51.012 --> 00:14:53.000
that's what that RAD 51 protein is doing.

bb060b51-36b6-4ff1-a31c-a1cc53010b9d-0
00:14:53.000 --> 00:14:54.280
And somehow it finds it.

1cc2ca93-4b5e-4b91-91a6-1098b325e3d3-0
00:14:55.520 --> 00:15:00.988
And so when it finds that donor template,
a structure called a displacement loop or

1cc2ca93-4b5e-4b91-91a6-1098b325e3d3-1
00:15:00.988 --> 00:15:02.160
AD loop is formed.

456c70b2-a2c1-4155-9011-968352b61429-0
00:15:02.360 --> 00:15:06.504
And So what that means is that it you,
if you think about DNA replication and

456c70b2-a2c1-4155-9011-968352b61429-1
00:15:06.504 --> 00:15:09.480
you have those two DNA strands,
it sort of opens it up.

19a45f74-cb9b-4d28-82c1-831cc4429180-0
00:15:09.600 --> 00:15:16.520
And so this donor template is disrupted
for a time being and it's used for repair.

82e2dc6d-d31e-4d89-a729-d696c438aae3-0
00:15:17.320 --> 00:15:22.235
And so the fifth step is new DNA
synthesis that will occur in a 5 prime to

82e2dc6d-d31e-4d89-a729-d696c438aae3-1
00:15:22.235 --> 00:15:27.086
three prime manner where you can see that
these little dots over here are

82e2dc6d-d31e-4d89-a729-d696c438aae3-2
00:15:27.086 --> 00:15:30.560
indicative of of new sequences being
copied on over.

86adc2aa-1f35-4a33-a08b-4056fd6bd373-0
00:15:31.240 --> 00:15:35.713
And so this intermediate right here is
that important step that I mentioned of

86adc2aa-1f35-4a33-a08b-4056fd6bd373-1
00:15:35.713 --> 00:15:39.677
sort of switching or replacement of
sequences where you can see we're

86adc2aa-1f35-4a33-a08b-4056fd6bd373-2
00:15:39.677 --> 00:15:43.754
switching from red sequences,
a mat A cell over to these blue sequences

86adc2aa-1f35-4a33-a08b-4056fd6bd373-3
00:15:43.754 --> 00:15:45.680
which contain mat alpha sequences.

ad19c59d-01f7-4b11-8ab4-5916ddc72319-0
00:15:46.400 --> 00:15:50.637
And so what's important here is that you
can see that this red sequence right over

ad19c59d-01f7-4b11-8ab4-5916ddc72319-1
00:15:50.637 --> 00:15:52.680
here is considered to be non homologous.

991f8f5d-2286-4a74-be0d-a12549541254-0
00:15:52.680 --> 00:15:54.080
So it needs to be removed.

78a4b7ae-8c78-43a0-bf38-fb097fcb8e3e-0
00:15:54.080 --> 00:15:56.880
So there are exonucleases that will come
and remove that.

0d968eaa-97fd-465e-871d-bd66f1ee260a-0
00:15:58.280 --> 00:16:03.014
And then the 6th step is that
displacement loop getting resolved and

0d968eaa-97fd-465e-871d-bd66f1ee260a-1
00:16:03.014 --> 00:16:06.720
that donor template will remain
completely unchanged.

44eac7f1-f588-46a8-954d-82728a3749fb-0
00:16:06.720 --> 00:16:10.270
So despite it getting disrupted,
those complementary strands getting

44eac7f1-f588-46a8-954d-82728a3749fb-1
00:16:10.270 --> 00:16:13.306
removed from each other,
nothing will happen to that donor

44eac7f1-f588-46a8-954d-82728a3749fb-2
00:16:13.306 --> 00:16:16.240
template after the correct sequences are
copied on over.

cf5be926-46aa-48bd-9b09-92186cf6de6f-0
00:16:17.320 --> 00:16:22.202
And then second strand synthesis happens
where you can see I sort of should have

cf5be926-46aa-48bd-9b09-92186cf6de6f-1
00:16:22.202 --> 00:16:26.120
made it a little bit longer,
but these are homologous sequences.

76e36586-7b8b-49a8-986a-d03ed6ff599e-0
00:16:26.120 --> 00:16:30.353
So they will pair up with each other and
then DNA synthesis will continue on over

76e36586-7b8b-49a8-986a-d03ed6ff599e-1
00:16:30.353 --> 00:16:32.160
in a 5 prime to three prime manner.

d131270f-70a5-448f-aefc-b692b686a648-0
00:16:33.760 --> 00:16:37.097
So then in the end,
we're left with newly synthesized DNA on

d131270f-70a5-448f-aefc-b692b686a648-1
00:16:37.097 --> 00:16:40.600
the recipient strand and that donor
template remains unchanged.

9d83008a-04c4-4c4b-b63a-97d6c8ebd262-0
00:16:40.600 --> 00:16:44.621
So you can see now that we have switched
on over from a matte A cell to a matte

9d83008a-04c4-4c4b-b63a-97d6c8ebd262-1
00:16:44.621 --> 00:16:48.240
alpha cell all through that just
switching or replacement of sequences.

cc03e641-345a-42ed-80be-90f8d026dd55-0
00:16:49.640 --> 00:16:53.930
And what I hope that you can all
appreciate is just how choreographed this

cc03e641-345a-42ed-80be-90f8d026dd55-1
00:16:53.930 --> 00:16:54.560
process is.

b0b078bd-5b81-48cf-ab44-ed3b485e61d2-0
00:16:55.200 --> 00:16:57.538
And I,
I failed to mention several like steps

b0b078bd-5b81-48cf-ab44-ed3b485e61d2-1
00:16:57.538 --> 00:17:00.080
only for the lack of time,
to be honest with you.

a62c7863-941f-4c79-b5c2-e198146c70b2-0
00:17:00.080 --> 00:17:02.440
But if you have any questions,
please let me know.

829b2778-8de4-4e21-bc04-2c10a53afe8c-0
00:17:03.600 --> 00:17:08.348
But this has been decades of research
both by Jim Haber and members in his lab,

829b2778-8de4-4e21-bc04-2c10a53afe8c-1
00:17:08.348 --> 00:17:10.960
as well as other labs all across the
world.

75e8d498-70b3-4c65-8c6d-15082337c5df-0
00:17:11.720 --> 00:17:16.849
And so this is a really nice experiment
that was done back in 1988 by Jim Haber

75e8d498-70b3-4c65-8c6d-15082337c5df-1
00:17:16.849 --> 00:17:20.120
and two other amazing scientists that are
up here.

bcabdb8d-5b8c-4720-9be0-0dfaabe5288b-0
00:17:21.480 --> 00:17:25.438
And So what they did was they were able
to physically monitor that mating type

bcabdb8d-5b8c-4720-9be0-0dfaabe5288b-1
00:17:25.438 --> 00:17:26.240
switching event.

f349cb52-db4b-4f84-ba59-e336929c989e-0
00:17:26.400 --> 00:17:29.320
So they were able to see the replacement
of those sequences.

1c1af9f6-3352-4595-a067-a803e3a919c2-0
00:17:29.920 --> 00:17:34.040
And what they did was a Southern blot
analysis or adna analysis.

4f5892d8-868a-41b8-821e-ded004137167-0
00:17:34.280 --> 00:17:39.000
And all that means is that you can sort
of see certain DNA fragments of interest.

6be5edac-bc08-4209-98fd-a6e44b1b0341-0
00:17:39.000 --> 00:17:43.925
And So what this does is it makes use of
restriction enzymes that are found all

6be5edac-bc08-4209-98fd-a6e44b1b0341-1
00:17:43.925 --> 00:17:45.280
throughout the genome.

bf983b3c-3d28-4e3a-9410-24ad7be74049-0
00:17:45.400 --> 00:17:47.120
And so that's what this S stands for.

04ca3074-1bd1-4d19-8567-5d66239fd565-0
00:17:47.120 --> 00:17:51.440
It's a specific restriction enzyme that
will cleave off this segment of DNA.

1400b0bb-91e5-4840-b4c7-7a61ec30a5fc-0
00:17:51.720 --> 00:17:54.496
And then what you can do is you can use
probes,

1400b0bb-91e5-4840-b4c7-7a61ec30a5fc-1
00:17:54.496 --> 00:17:59.240
which are labeled DNA fragments that can
identify specific sequences in a sample.

2b65d38f-92ab-4e27-b1cf-a65755fca1b4-0
00:17:59.800 --> 00:18:02.160
And so here what we're looking at is time.

55188435-a755-425f-b629-67b79814bb8d-0
00:18:02.400 --> 00:18:05.920
So you can 012 and four hours.

37ad4b10-fd5b-468e-ac1b-4a73c33505c1-0
00:18:06.440 --> 00:18:11.726
And so here we're looking at a MAT A cell
at 0 and what they have done is they have

37ad4b10-fd5b-468e-ac1b-4a73c33505c1-1
00:18:11.726 --> 00:18:15.440
added that galactose,
so they have turned on that HO gene.

7cae0596-8daa-4335-adc8-480643737bd9-0
00:18:15.520 --> 00:18:19.418
So therefore that HO endonuclease can
come and cleave right over here,

7cae0596-8daa-4335-adc8-480643737bd9-1
00:18:19.418 --> 00:18:21.560
this galactose induced HO endonuclease.

722f5120-e375-48dd-96d2-d1659c787014-0
00:18:21.960 --> 00:18:27.136
And what you can see here is that there
is cut DNA and then after some time there

722f5120-e375-48dd-96d2-d1659c787014-1
00:18:27.136 --> 00:18:30.040
is new DNA found and that is a MAT alpha
DNA.

d894194a-28ac-455c-bb35-7321cde8dfde-0
00:18:30.040 --> 00:18:32.460
And you could start to see it a little
bit at two hours,

d894194a-28ac-455c-bb35-7321cde8dfde-1
00:18:32.460 --> 00:18:34.880
but you could really distinctly see it
after four hours.

650948b7-92ac-4be8-8e05-af376fbc4004-0
00:18:36.200 --> 00:18:38.855
OK,
so I'm going to switch gears a little bit

650948b7-92ac-4be8-8e05-af376fbc4004-1
00:18:38.855 --> 00:18:41.280
now and talk a little bit about chromatin.

48a4c669-a59a-4558-93b8-02cdc9f93e83-0
00:18:41.920 --> 00:18:49.458
So if we were to decondense a chromosome,
we will find that there is chromatin,

48a4c669-a59a-4558-93b8-02cdc9f93e83-1
00:18:49.458 --> 00:18:56.997
which is a complex of DNA and proteins
that serves as the functional form of of

48a4c669-a59a-4558-93b8-02cdc9f93e83-2
00:18:56.997 --> 00:18:58.600
genetic material.

da6e1551-d0cc-409a-8739-2c5332aef543-0
00:18:58.800 --> 00:19:03.796
And this allows for a way to for a cell
to compact all of its DNA in an efficient

da6e1551-d0cc-409a-8739-2c5332aef543-1
00:19:03.796 --> 00:19:04.040
way.

3c30d989-b841-4065-bf78-c3bcacee3f4f-0
00:19:04.040 --> 00:19:06.520
But importantly,
it allows for gene regulation.

bd19a84c-31a1-485c-a087-4371d258d0ce-0
00:19:08.160 --> 00:19:11.752
And so the basic unit of chromatin
consists of DNA wrapped around these

bd19a84c-31a1-485c-a087-4371d258d0ce-1
00:19:11.752 --> 00:19:13.000
proteins called histones.

ebc5ee9c-5c7f-478a-ab8a-9cf2f8e44215-0
00:19:14.200 --> 00:19:16.560
And it's specifically wrapped around a
histone octomer.

86fd6314-bb9c-44c9-bd56-1bdbb49054cd-0
00:19:17.680 --> 00:19:22.400
And it's what's needed for about 150 base
pairs or so of DNA to wrap around.

4f58e5b7-6e2d-48c6-8b80-2acccc329368-0
00:19:23.080 --> 00:19:26.200
And so you can see that the histone
octomer makes up it.

f804fd12-9081-44f2-90d2-be19fed78f73-0
00:19:26.280 --> 00:19:30.760
It makes up two copies of H2BH2AH3 and H4.

429ee549-2017-4449-a547-8cc25e04bad5-0
00:19:31.680 --> 00:19:35.274
And what's important is it can form this
structure called a nucleosome,

429ee549-2017-4449-a547-8cc25e04bad5-1
00:19:35.274 --> 00:19:37.920
which is just the basic repeating unit of
chromatin.

b1edf528-35ca-4c7b-97cd-0eeab7707cfa-0
00:19:39.640 --> 00:19:43.162
And so in eukaryotes,
there are two chromatin structures that

b1edf528-35ca-4c7b-97cd-0eeab7707cfa-1
00:19:43.162 --> 00:19:45.720
exist called eukromatin and
heterochromatin.

460bf77d-0e4b-4414-b492-a1cb5592334a-0
00:19:46.320 --> 00:19:51.137
So eukromatin is characterized as loosely
packed transcriptionally active regions

460bf77d-0e4b-4414-b492-a1cb5592334a-1
00:19:51.137 --> 00:19:51.960
in the genome.

20c6a0f8-b2d5-46d5-b6a6-8226bcaed053-0
00:19:52.560 --> 00:19:55.400
So the DNA is accessible and that gene is
active.

3453a995-1e33-44d9-b761-509d375589e8-0
00:19:55.960 --> 00:19:59.003
In contrast,
heterochromatin is characterized as

3453a995-1e33-44d9-b761-509d375589e8-1
00:19:59.003 --> 00:20:02.605
densely packed,
transcriptionally inactive regions in the

3453a995-1e33-44d9-b761-509d375589e8-2
00:20:02.605 --> 00:20:03.040
genome.

ec01a64a-cf1e-4cb2-b608-c77d57958315-0
00:20:03.040 --> 00:20:06.280
So therefore that DNA is inaccessible and
that gene is active.

6c19ae2e-e52f-4dcb-9646-122061fbc14d-0
00:20:09.880 --> 00:20:13.774
In budding yeast,
there are thought to be 3 major regions

6c19ae2e-e52f-4dcb-9646-122061fbc14d-1
00:20:13.774 --> 00:20:17.400
of transcriptionally silent or
heterochromatic areas.

c053ed85-d81b-499d-971e-6986f75ff935-0
00:20:17.680 --> 00:20:21.848
I'm not going to talk about these two
today because what's really of interest

c053ed85-d81b-499d-971e-6986f75ff935-1
00:20:21.848 --> 00:20:23.880
to me is that silent mating type loci.

a0af4dda-6eec-486b-9a95-619adacf16f8-0
00:20:25.440 --> 00:20:29.489
And so let's look a little bit closer at
what's really of interest to me,

a0af4dda-6eec-486b-9a95-619adacf16f8-1
00:20:29.489 --> 00:20:32.280
which is that HML heterochromatic donor
over here.

a94c1576-2fd3-428c-a20c-4e8556512611-0
00:20:32.960 --> 00:20:35.890
And so I already showed you this genetic
arrangement earlier,

a94c1576-2fd3-428c-a20c-4e8556512611-1
00:20:35.890 --> 00:20:39.720
but I'm just going to highlight a few
different things that I didn't previously.

8e5a7481-7b4f-409d-a017-6a160a25ce84-0
00:20:40.080 --> 00:20:45.870
So there are three things that are really
important for this HML locus to become

8e5a7481-7b4f-409d-a017-6a160a25ce84-1
00:20:45.870 --> 00:20:47.800
transcriptionally inactive.

937373e4-e25a-499c-9480-9e2758117b9c-0
00:20:48.320 --> 00:20:53.512
The first is these E and I silencers,
and so these encode sequences that DNA

937373e4-e25a-499c-9480-9e2758117b9c-1
00:20:53.512 --> 00:20:56.480
binding proteins can recognize and bind
to.

949728f2-5703-490c-91c4-a3203d61edce-0
00:20:57.640 --> 00:21:04.013
I honestly forgot what E stands for,
but I stands for important for silencing

949728f2-5703-490c-91c4-a3203d61edce-1
00:21:04.013 --> 00:21:07.200
and so this was when it was discovered.

5843d3f6-67d7-46c7-9578-9ab8e9e9990e-0
00:21:07.760 --> 00:21:10.104
However,
you can see that it contains the same

5843d3f6-67d7-46c7-9578-9ab8e9e9990e-1
00:21:10.104 --> 00:21:12.200
genetic arrangement as a matte alpha cell.

0a59174e-9ebe-46df-a12f-cbae51fceae9-0
00:21:12.760 --> 00:21:15.280
So it's WXY and Z.

d0704118-51aa-44ff-84d5-4bc980c6511b-0
00:21:16.600 --> 00:21:20.360
And so the second thing that's important
for silencing at HML are those DNA

d0704118-51aa-44ff-84d5-4bc980c6511b-1
00:21:20.360 --> 00:21:23.280
binding proteins that can bind to those E
and I silencers.

807c624c-2ee5-4712-b216-48da556f11a3-0
00:21:23.920 --> 00:21:27.837
And then the third thing are well
positioned nucleosomes across this HML

807c624c-2ee5-4712-b216-48da556f11a3-1
00:21:27.837 --> 00:21:28.160
locus.

8e6fe0bc-27c9-4a3d-9d9a-0b47f22d5cfe-0
00:21:29.080 --> 00:21:34.360
And so this is an experiment that was
done way back in 1998.

0a6380f1-2ed4-4c9d-83d1-5155f319ad19-0
00:21:35.080 --> 00:21:40.600
And So what they were able to do is map
the histones across HML.

c15c2b06-8ceb-4878-a035-f80523c0b175-0
00:21:40.880 --> 00:21:44.760
And so you can see here that there are
some well positioned nucleosomes.

72320274-3292-49a4-8aaf-b3980463497b-0
00:21:44.880 --> 00:21:46.930
However,
there's also some less defined

72320274-3292-49a4-8aaf-b3980463497b-1
00:21:46.930 --> 00:21:48.160
nucleosomes around here.

5e530176-d430-40c7-b6bb-3aa1b9a99630-0
00:21:48.360 --> 00:21:53.378
But what's important here is that across
the the regions that are really important

5e530176-d430-40c7-b6bb-3aa1b9a99630-1
00:21:53.378 --> 00:21:56.704
for silencing,
specifically sort of where this HO endo

5e530176-d430-40c7-b6bb-3aa1b9a99630-2
00:21:56.704 --> 00:22:00.635
nucleus can recognize,
there are well positioned histones around

5e530176-d430-40c7-b6bb-3aa1b9a99630-3
00:22:00.635 --> 00:22:01.239
that area.

4f1020ca-7efc-4e98-a1bf-4e01b4f53e87-0
00:22:04.120 --> 00:22:09.440
So here we're looking at a zoomed in
picture of this E silencer.

67ff0f36-af0c-46ef-8823-72d51b01d924-0
00:22:09.520 --> 00:22:11.480
So from here all the way to here.

836f8c33-d555-4752-9cb8-bbef4f5ce185-0
00:22:11.880 --> 00:22:16.140
And what I'm going to talk about now is
sort of how that heterochromatin

836f8c33-d555-4752-9cb8-bbef4f5ce185-1
00:22:16.140 --> 00:22:17.600
structure is established.

3149c0fc-34cc-47a6-b5a0-4baa50bfd480-0
00:22:18.280 --> 00:22:22.389
So I already told you that that E
silencer encodes 4 sequences where DNA

3149c0fc-34cc-47a6-b5a0-4baa50bfd480-1
00:22:22.389 --> 00:22:24.360
binding proteins can come and bind.

701ee70c-b820-46b8-9c88-27932e069cd8-0
00:22:24.880 --> 00:22:28.480
So that's what these 3 grey circles
indicate.

d4185cc2-bf11-42c2-9c86-123f822e250e-0
00:22:29.040 --> 00:22:33.469
And these DNA binding proteins can
recruit another set of proteins that are

d4185cc2-bf11-42c2-9c86-123f822e250e-1
00:22:33.469 --> 00:22:36.208
very,
very important for silencing and they're

d4185cc2-bf11-42c2-9c86-123f822e250e-2
00:22:36.208 --> 00:22:39.880
called silent information regulatory
proteins or SER proteins.

93a28ebd-4b18-404f-830d-faefb0992883-0
00:22:40.680 --> 00:22:43.520
And so there are four different SER
proteins.

6d781c5d-d370-4cd6-b895-9f9ce72e1b2d-0
00:22:43.640 --> 00:22:48.502
And so one of these DNA binding proteins
will recruit SER one,

6d781c5d-d370-4cd6-b895-9f9ce72e1b2d-1
00:22:48.502 --> 00:22:53.520
which will then recruit SER 4,
SER 3 and then importantly SER 2.

a0833762-bc7e-4e91-bc7d-fa13006907c9-0
00:22:54.160 --> 00:22:56.760
Now SER 2 is a histone deacetylase.

6b356e26-9942-4bcc-88cb-5745a632e98e-0
00:22:56.960 --> 00:23:01.767
And So what that means is that it can
catalyze a reaction where it can remove

6b356e26-9942-4bcc-88cb-5745a632e98e-1
00:23:01.767 --> 00:23:03.000
an acetylation mark.

c37834e4-e822-4f60-b595-f4f3d5c42de3-0
00:23:03.360 --> 00:23:08.260
And so specifically on these well
positioned histones across HML,

c37834e4-e822-4f60-b595-f4f3d5c42de3-1
00:23:08.260 --> 00:23:13.310
there are many post translational
modifications, but what's really,

c37834e4-e822-4f60-b595-f4f3d5c42de3-2
00:23:13.310 --> 00:23:17.840
really important for silencing is this
H3KH4K16 acetylation.

0564d418-2f9a-44d3-b980-a5ed3c9b7e01-0
00:23:18.960 --> 00:23:22.540
And for those of you that don't speak
this language,

0564d418-2f9a-44d3-b980-a5ed3c9b7e01-1
00:23:22.540 --> 00:23:27.134
what this means is that this H4 protein
on its 16th lysine residue,

0564d418-2f9a-44d3-b980-a5ed3c9b7e01-2
00:23:27.134 --> 00:23:31.120
there's an acetylation modification that
was placed there.

8b0e69d3-695e-4db7-bc10-e8d10d0ad4c0-0
00:23:31.800 --> 00:23:35.760
And so Sir Two, what it will do,
it was it will recognize this specific

8b0e69d3-695e-4db7-bc10-e8d10d0ad4c0-1
00:23:35.760 --> 00:23:37.960
modification and it will deacetylate it.

9ca4f868-2606-4f78-abcd-366bd1b82240-0
00:23:38.680 --> 00:23:42.328
And what this allows for is Sir 3 to come
and bind,

9ca4f868-2606-4f78-abcd-366bd1b82240-1
00:23:42.328 --> 00:23:47.800
which will then recruit Sir 2 and then
continue on in a subsequential manner.

894b4efc-df2c-4f4b-9a7c-e0cb2bf7b74a-0
00:23:47.840 --> 00:23:50.160
We'll recruit Sir 3 and so forth.

f47d2b16-f53b-4cee-b4f8-98e9a948feb0-0
00:23:50.360 --> 00:23:54.620
And So what you can start to see is sort
of how this heterochromatic structure is

f47d2b16-f53b-4cee-b4f8-98e9a948feb0-1
00:23:54.620 --> 00:23:55.400
is established.

b2db0005-1611-4c52-b297-e7c41035197a-0
00:23:58.000 --> 00:24:00.120
And so this is a very stable structure.

2ecb8fc1-541c-4cd1-baf9-719d4ead6842-0
00:24:01.680 --> 00:24:08.689
And so there there's a lab over at UC
Berkeley called Jasper Rhines Lab where

2ecb8fc1-541c-4cd1-baf9-719d4ead6842-1
00:24:08.689 --> 00:24:15.520
they have developed this genetic assay to
measure loss of silencing at HML.

9e5c4419-3d2f-4908-a2ad-ae886a98eafc-0
00:24:15.720 --> 00:24:21.080
So they wanted to see well how stable is
that heterochromatic structure at HML.

fc0a0a54-cf2a-4c18-b5b6-757f094c8743-0
00:24:21.520 --> 00:24:27.137
And so they did something very clever
where they made use of genes that are

fc0a0a54-cf2a-4c18-b5b6-757f094c8743-1
00:24:27.137 --> 00:24:28.320
found here at Y.

247e73c9-dc24-4f4c-a9ec-6b189b3c1b18-0
00:24:28.600 --> 00:24:34.056
And what I failed to mention earlier
about those genes that are encoded within

247e73c9-dc24-4f4c-a9ec-6b189b3c1b18-1
00:24:34.056 --> 00:24:38.408
MAT A or MAT alpha,
which are also found again at HML and HMR,

247e73c9-dc24-4f4c-a9ec-6b189b3c1b18-2
00:24:38.408 --> 00:24:42.000
is that there are two of them called Y A2
and Y A1.

8b69f01e-84c4-4a8f-b1db-6b4f5b2d4391-0
00:24:42.760 --> 00:24:47.390
And So what they did was they simply
replaced that Y A2 gene with the Cree

8b69f01e-84c4-4a8f-b1db-6b4f5b2d4391-1
00:24:47.390 --> 00:24:48.440
recombinase gene.

f83063e9-a299-4708-9dcc-3c7ec1d18901-0
00:24:49.760 --> 00:24:52.560
And so this is again a heterochromatic
structure.

8019afec-8f05-4910-9838-06df90612f70-0
00:24:52.920 --> 00:24:57.989
And So what this allows for us to do is
basically see if that create gene is ever

8019afec-8f05-4910-9838-06df90612f70-1
00:24:57.989 --> 00:25:00.956
expressed,
it will mediate the recombination of

8019afec-8f05-4910-9838-06df90612f70-2
00:25:00.956 --> 00:25:02.440
these two locks P sites.

22815737-7a87-42c4-8f43-395be0fedd26-0
00:25:02.600 --> 00:25:07.057
So the Cree recombinase protein is an
enzyme that can recognize these locks P

22815737-7a87-42c4-8f43-395be0fedd26-1
00:25:07.057 --> 00:25:07.400
sites.

3163c44f-ca50-4097-84ac-794cb6ffd59a-0
00:25:07.640 --> 00:25:11.842
And this is a really nice genetic tool
because what it does is you can use these

3163c44f-ca50-4097-84ac-794cb6ffd59a-1
00:25:11.842 --> 00:25:14.800
locks P sites and sort of Orient them in
different ways.

5ffcb679-2714-4616-af39-4ce741afded7-0
00:25:15.000 --> 00:25:18.119
This has been studies that were done many
decades ago,

5ffcb679-2714-4616-af39-4ce741afded7-1
00:25:18.119 --> 00:25:22.713
and essentially what they have done here
is they've oriented them in a way where

5ffcb679-2714-4616-af39-4ce741afded7-2
00:25:22.713 --> 00:25:26.344
if Cree is ever expressed,
it will mediate the recombination of

5ffcb679-2714-4616-af39-4ce741afded7-3
00:25:26.344 --> 00:25:31.051
these two locks piece sites and therefore
excise anything that is found in between

5ffcb679-2714-4616-af39-4ce741afded7-4
00:25:31.051 --> 00:25:32.640
these two locks piece sites.

97fd67f2-a1ba-46a9-bf6c-4057dec84184-0
00:25:32.800 --> 00:25:38.440
So what you can see here is that you have
a cell that has all these genes here.

fc331cfd-0430-475c-9fc7-af14b9fd3ef8-0
00:25:38.560 --> 00:25:42.040
However, if there's a loss of silencing,
you will lose these two genes.

571b44fb-26df-4b56-b66a-1ded63951f14-0
00:25:42.400 --> 00:25:46.923
And So what this does is that if a cell
has that heterochromatic structure

571b44fb-26df-4b56-b66a-1ded63951f14-1
00:25:46.923 --> 00:25:50.120
maintained, meaning it is stable,
cells will be red.

b9927973-e14b-41d5-b011-99bb8ffa0edb-0
00:25:50.280 --> 00:25:54.345
So if I look under a fluorescent
microscope, I will see that they are red,

b9927973-e14b-41d5-b011-99bb8ffa0edb-1
00:25:54.345 --> 00:25:58.411
and that is because they have this RFP
gene and that encodes for something

b9927973-e14b-41d5-b011-99bb8ffa0edb-2
00:25:58.411 --> 00:26:00.200
called a red fluorescent protein.

dc1178ac-beee-4276-87f0-87386b9cb54f-0
00:26:00.960 --> 00:26:05.109
Additionally,
they are resistant to this drug called G4

dc1178ac-beee-4276-87f0-87386b9cb54f-1
00:26:05.109 --> 00:26:05.480
O 18.

3126fd8c-77d9-4eff-928c-6a9782e459fd-0
00:26:06.120 --> 00:26:09.539
And this is just really nice in the lab
where you can just do a bunch of

3126fd8c-77d9-4eff-928c-6a9782e459fd-1
00:26:09.539 --> 00:26:11.320
different things with different media.

cbcddf6e-6ad9-45ed-b01b-575ffb916378-0
00:26:12.800 --> 00:26:18.493
And So what this does is it allows for us
to visually see a cell go from red to

cbcddf6e-6ad9-45ed-b01b-575ffb916378-1
00:26:18.493 --> 00:26:18.920
green.

8cdf470f-5e6e-4ff6-949c-72b7457593af-0
00:26:19.600 --> 00:26:24.973
And so for those of you that don't know
sort of how yeast lab works,

8cdf470f-5e6e-4ff6-949c-72b7457593af-1
00:26:24.973 --> 00:26:28.400
this is also true for many other
organisms.

e11eef4c-0dec-4f5d-b150-45ff9c34fa2e-0
00:26:28.400 --> 00:26:33.233
But what we can do is we can take an Agar
plate,

e11eef4c-0dec-4f5d-b150-45ff9c34fa2e-1
00:26:33.233 --> 00:26:38.560
which is essentially a sort of gel
nutritional media.

8f4159a9-5e55-46cf-8dc2-a1751c59478e-0
00:26:39.000 --> 00:26:43.571
And what I can do is I can take a single
cell and I can plate it on that Agar

8f4159a9-5e55-46cf-8dc2-a1751c59478e-1
00:26:43.571 --> 00:26:46.560
plate and I can wait maybe like two or
three days.

50db0eae-04e8-4879-b40e-0fa60688de74-0
00:26:46.920 --> 00:26:50.560
And what I will be able to see is
visually a colony.

6350ce14-422e-49ee-9300-80e181162c2c-0
00:26:50.560 --> 00:26:53.403
And what that is,
is a bunch of different cells that have

6350ce14-422e-49ee-9300-80e181162c2c-1
00:26:53.403 --> 00:26:55.120
grown up for me to visually see it.

e88c4149-f1af-4605-88b0-3577dccfe15f-0
00:26:56.000 --> 00:27:01.080
And so here is a colony of a single cell
where you can see it.

45e3e831-3572-40f9-a239-eeacb74ea247-0
00:27:01.080 --> 00:27:04.725
It is,
it is red and it has this little sort of

45e3e831-3572-40f9-a239-eeacb74ea247-1
00:27:04.725 --> 00:27:07.080
stripe over here that is green.

e0ba3d4c-adee-4539-af23-6420f4ad876b-0
00:27:07.080 --> 00:27:11.374
And so that means that like some cells
that have sort of formed to make this

e0ba3d4c-adee-4539-af23-6420f4ad876b-1
00:27:11.374 --> 00:27:12.880
colony have lost silencing.

fd33090e-cae7-4041-ba75-0d4e01242cd4-0
00:27:13.520 --> 00:27:19.852
And what was novel about sort of using
this genetic tool is that about one to 2%

fd33090e-cae7-4041-ba75-0d4e01242cd4-1
00:27:19.852 --> 00:27:23.840
of cells lose silencing during normal
replication.

65a2ca0e-88df-4c93-93c3-3715fcdd43d2-0
00:27:24.080 --> 00:27:28.448
So what that means is that some cells
that have grown up into that colony seem

65a2ca0e-88df-4c93-93c3-3715fcdd43d2-1
00:27:28.448 --> 00:27:29.720
to have lost silencing.

e0904370-8bbf-40ea-b15a-6404ab90513b-0
00:27:29.720 --> 00:27:33.799
And it gave this phenotype over here of
half green and half red,

e0904370-8bbf-40ea-b15a-6404ab90513b-1
00:27:33.799 --> 00:27:38.757
because what this means is that either
the first or second cell that gave rise

e0904370-8bbf-40ea-b15a-6404ab90513b-2
00:27:38.757 --> 00:27:40.639
to that colony lost silencing.

e690e2e5-fa3b-446b-8d09-c4ba800c97ad-0
00:27:43.360 --> 00:27:48.655
So this brings me to sort of my research
project and what I'm really interested in,

e690e2e5-fa3b-446b-8d09-c4ba800c97ad-1
00:27:48.655 --> 00:27:51.869
which is to sort of answer this question
of, well,

e690e2e5-fa3b-446b-8d09-c4ba800c97ad-2
00:27:51.869 --> 00:27:56.597
how is the stability of heterochromatin
maintenance affected by homologous

e690e2e5-fa3b-446b-8d09-c4ba800c97ad-3
00:27:56.597 --> 00:27:57.480
recombination?

3fd0e672-b830-4005-bacf-f76a8ec92c4b-0
00:27:57.680 --> 00:27:59.880
And broadly,
how is that regulated in the cell?

2c662520-60c5-43fc-91e2-0048379cf940-0
00:28:00.480 --> 00:28:05.220
And So what I can do is I can use that
crash assay and modify it to do several

2c662520-60c5-43fc-91e2-0048379cf940-1
00:28:05.220 --> 00:28:06.240
different things.

300ac497-d9a7-4a15-b18c-447dc6e47be4-0
00:28:06.840 --> 00:28:11.441
And So what I'm really interested in is
this intermediate right over here where I

300ac497-d9a7-4a15-b18c-447dc6e47be4-1
00:28:11.441 --> 00:28:14.360
told you that that displacement loop can
be formed.

4ced31e7-da05-4a4d-add4-b9c698412cf8-0
00:28:14.960 --> 00:28:19.878
I'm really interested to understand how
exactly does that donor template open up

4ced31e7-da05-4a4d-add4-b9c698412cf8-1
00:28:19.878 --> 00:28:21.640
and allow for that to happen.

54227ede-48d6-4917-99e0-e762342f0749-0
00:28:21.760 --> 00:28:25.517
Since it is a heterochromatic structure
and it is stable,

54227ede-48d6-4917-99e0-e762342f0749-1
00:28:25.517 --> 00:28:30.960
how exactly does that change to allow for
repair to happen and why does it do that?

de558b7d-dffe-4426-bc0a-0f408750baa1-0
00:28:31.080 --> 00:28:34.920
Why does it do that in in this context
but not in other contexts?

ea1d04f8-c6b5-43d7-a961-1ab39aede7b9-0
00:28:36.320 --> 00:28:42.447
And So what we have done is we have
constructed this pseudo map,

ea1d04f8-c6b5-43d7-a961-1ab39aede7b9-1
00:28:42.447 --> 00:28:48.480
a fragment over here and inserted it on a
different chromosome.

b55229c4-ef6c-46ab-8920-1e4510725bd8-0
00:28:48.480 --> 00:28:51.320
So you can see I've inserted it into
chromosome 8.

3d7f610f-6166-44b4-8df1-202bc2286b30-0
00:28:52.040 --> 00:28:57.000
And So what this contains is a truncated
Cree gene right over here.

f432b373-008b-42a2-a82b-27b0ba472272-0
00:28:57.280 --> 00:29:01.427
And So what this allows for is just the
reporter system to to work because if you

f432b373-008b-42a2-a82b-27b0ba472272-1
00:29:01.427 --> 00:29:03.854
can imagine that Cree gene is always
expressed,

f432b373-008b-42a2-a82b-27b0ba472272-2
00:29:03.854 --> 00:29:06.080
that means that cells will always be
green.

ba5eb44d-682c-4677-95d2-c55db58d1904-0
00:29:06.160 --> 00:29:09.680
So it doesn't give us a measurement,
a readout of loss of silencing.

515d1923-87dd-4acc-994e-c252030f1fa1-0
00:29:10.960 --> 00:29:14.567
Additionally, it contains that Y A1 gene,
this Z,

515d1923-87dd-4acc-994e-c252030f1fa1-1
00:29:14.567 --> 00:29:19.040
and then a little bit of that HML and in
total it makes up 2.

515d1923-87dd-4acc-994e-c252030f1fa1-2
00:29:19.040 --> 00:29:22.720
8 kilobases of homology between pseudomat
and HML.

d3320f5b-853d-4d15-bc16-319acf3625d7-0
00:29:23.640 --> 00:29:28.890
And So what I can do is I can use that
Galicho system to conditionally induce

d3320f5b-853d-4d15-bc16-319acf3625d7-1
00:29:28.890 --> 00:29:34.611
adna double strand break at the pseudomat
locus and then I can ask a question, well,

d3320f5b-853d-4d15-bc16-319acf3625d7-2
00:29:34.611 --> 00:29:38.920
what happens to silencing after I induce
a double strand break?

949eebf3-c359-4536-8579-1dbb2979319d-0
00:29:41.320 --> 00:29:47.988
So what I find is that about 50% of cells
experience a loss of silencing event at

949eebf3-c359-4536-8579-1dbb2979319d-1
00:29:47.988 --> 00:29:48.720
HML Cree.

646eca99-ea3a-4b4b-a235-b191b3f8e6aa-0
00:29:49.800 --> 00:29:53.280
And specifically this is about after four
hours.

a2ccea92-0189-45b5-bb37-7b5054443fad-0
00:29:53.560 --> 00:29:57.991
So here we're looking at 0 where I didn't
add galactose and then this is half an

a2ccea92-0189-45b5-bb37-7b5054443fad-1
00:29:57.991 --> 00:30:01.329
hour all the way to 4 hours after adding
that galactose and,

a2ccea92-0189-45b5-bb37-7b5054443fad-2
00:30:01.329 --> 00:30:04.120
and conditionally turning on that HO
endonuclease.

2df85b87-6bc9-4912-8288-984f4a422cbd-0
00:30:04.880 --> 00:30:10.520
And so this brings up a lot of questions,
which is, well, why is it 50% of the time?

ca63ecb1-e9b4-4154-b284-4ed4d6889b5e-0
00:30:10.520 --> 00:30:11.840
Why is it not more?

e6d48b91-419d-4289-b295-cb3c2ba40c21-0
00:30:11.840 --> 00:30:13.040
Why is it not less?

ae220d64-36ee-4b99-9517-926ba960c69d-0
00:30:14.240 --> 00:30:19.156
So there's a lot of intricacies to,
to work out since it's not simply adna

ae220d64-36ee-4b99-9517-926ba960c69d-1
00:30:19.156 --> 00:30:21.320
repair event that I'm looking at.

ad128f0a-4356-4e88-be75-8ab68da78117-0
00:30:21.480 --> 00:30:23.800
I'm also looking at a transcriptional
event, right?

a16fc32e-2801-4115-9797-375efd454b45-0
00:30:23.800 --> 00:30:27.280
I'm looking at a transcriptional event
from that HML locus itself.

d40232b6-4286-4c61-a74f-4e2fa7b0ffe1-0
00:30:28.800 --> 00:30:33.618
And so I'm going to tell you two things
that I am currently working on to work

d40232b6-4286-4c61-a74f-4e2fa7b0ffe1-1
00:30:33.618 --> 00:30:34.960
out those intricacies.

2e9e271b-23f5-4669-8e36-e7931925c9d9-0
00:30:36.400 --> 00:30:41.355
And so the first thing that I'm really
interested in is sort of assessing, well,

2e9e271b-23f5-4669-8e36-e7931925c9d9-1
00:30:41.355 --> 00:30:46.310
how much of an effect does the length of
homology have on that loss of silencing

2e9e271b-23f5-4669-8e36-e7931925c9d9-2
00:30:46.310 --> 00:30:46.800
outcome.

ee9c7bbd-e5a3-48f8-8c01-2b752b63e038-0
00:30:46.800 --> 00:30:49.320
So what I want to do is I want to
decrease that homology.

91655c0e-5adf-4834-87f8-be2d2a7da7be-0
00:30:49.520 --> 00:30:54.065
And so I want to delete this truncated
Cree over here and see what effect does

91655c0e-5adf-4834-87f8-be2d2a7da7be-1
00:30:54.065 --> 00:30:56.080
that have to the loss of silencing.

362698de-d7fe-4165-8d02-b9a995b3081a-0
00:30:56.320 --> 00:31:01.052
Since I told you earlier,
and I'm going to go back to sort of this

362698de-d7fe-4165-8d02-b9a995b3081a-1
00:31:01.052 --> 00:31:05.219
step right over here,
is that the really important part of

362698de-d7fe-4165-8d02-b9a995b3081a-2
00:31:05.219 --> 00:31:10.093
double strand brake repair is,
is sort of this copping over of those

362698de-d7fe-4165-8d02-b9a995b3081a-3
00:31:10.093 --> 00:31:10.800
sequences.

f80adfc4-727f-488e-b031-ed855f7340bd-0
00:31:10.920 --> 00:31:14.809
And what's really cool about mating type
switching is it sort of knows when to

f80adfc4-727f-488e-b031-ed855f7340bd-1
00:31:14.809 --> 00:31:15.400
stop almost.

7be4a3ec-9c59-4b36-aa82-c331585a5d2a-0
00:31:16.040 --> 00:31:20.853
And so I want to assess that in my own
system and see what will happen if I

7be4a3ec-9c59-4b36-aa82-c331585a5d2a-1
00:31:20.853 --> 00:31:22.120
delete that cregene.

4ae132e3-7d1d-492c-93c4-e369e4409bf2-0
00:31:22.200 --> 00:31:23.920
Well, how far does it go?

a35e399c-457d-48f0-8284-e296f8096ed9-0
00:31:24.160 --> 00:31:27.080
Will I get less loss of silencing
outcomes?

db3e40d3-fb25-4560-b5d1-dc058fdd3653-0
00:31:27.520 --> 00:31:28.920
Does it not make a difference?

df3b6066-a987-4a30-a484-6e6452a51259-0
00:31:29.160 --> 00:31:32.120
So this is just one intricacy that I'm
currently working on.

8492d026-6c5c-4f96-b5f8-799624e1108a-0
00:31:34.040 --> 00:31:38.789
The second thing is I want to design a
system that sort of mimics normal mating

8492d026-6c5c-4f96-b5f8-799624e1108a-1
00:31:38.789 --> 00:31:39.680
type switching.

4668bf96-d7c8-4928-88c9-ca906a9b38b1-0
00:31:40.200 --> 00:31:44.574
So here in this system,
we're not looking at a replacement of

4668bf96-d7c8-4928-88c9-ca906a9b38b1-1
00:31:44.574 --> 00:31:45.280
sequences.

aea76261-b452-46f3-a2a0-078e0d0b4df2-0
00:31:45.440 --> 00:31:48.880
It's just copying over the same sequences
that have been broken, right?

02edcc54-2e1a-4823-ad27-6f0abccd868f-0
00:31:49.160 --> 00:31:54.239
And So what I would like to do is I would
like to replace that A1 sequence with

02edcc54-2e1a-4823-ad27-6f0abccd868f-1
00:31:54.239 --> 00:31:58.430
that A1 sequence and assess what happens
to to loss of silencing,

02edcc54-2e1a-4823-ad27-6f0abccd868f-2
00:31:58.430 --> 00:32:02.240
since this will sort of mimic normal
mating type switching.

186a4348-a666-4f0b-948a-7530e9f64645-0
00:32:02.440 --> 00:32:06.479
And therefore we can sort of learn a
little bit more about mating type

186a4348-a666-4f0b-948a-7530e9f64645-1
00:32:06.479 --> 00:32:10.802
switching and add on to decades of
research that was done by both Jim Haber

186a4348-a666-4f0b-948a-7530e9f64645-2
00:32:10.802 --> 00:32:12.680
and many other labs in the world.

e6f5560d-3108-478f-a263-fc8a832556d3-0
00:32:14.040 --> 00:32:17.741
And so with that,
I would like to just quickly summarise

e6f5560d-3108-478f-a263-fc8a832556d3-1
00:32:17.741 --> 00:32:19.560
everything that I went over.

0bf3f4c8-563e-451a-9c37-d006e2e72dc2-0
00:32:20.480 --> 00:32:24.402
So mating type switching and budding
yeast depends on a a double strand break,

0bf3f4c8-563e-451a-9c37-d006e2e72dc2-1
00:32:24.402 --> 00:32:28.325
which is repaired through homologous
recombination using those heterochromatic

0bf3f4c8-563e-451a-9c37-d006e2e72dc2-2
00:32:28.325 --> 00:32:29.120
donor templates.

d4ba643c-00e4-4e91-978f-32d82d6a42fe-0
00:32:29.640 --> 00:32:32.080
There are many steps involved in mating
type switching.

505db26a-5aef-4b5b-b45c-0b0f6a4a0a21-0
00:32:32.520 --> 00:32:35.789
The dynamics of heterochromatin
establishment and maintenance can be

505db26a-5aef-4b5b-b45c-0b0f6a4a0a21-1
00:32:35.789 --> 00:32:37.400
measured through that crash assay.

44bb2157-6c51-4813-8d23-09b054212176-0
00:32:38.400 --> 00:32:42.690
After inducing a double strand break,
about 50% of cells experience a loss of

44bb2157-6c51-4813-8d23-09b054212176-1
00:32:42.690 --> 00:32:43.240
silencing.

41ff4947-f95c-46e4-8968-af86969705c0-0
00:32:43.800 --> 00:32:47.569
And then lastly,
my research questions I hope will provide

41ff4947-f95c-46e4-8968-af86969705c0-1
00:32:47.569 --> 00:32:52.680
insight into how chromatin structure
effects recombination and gene expression.

b99f42c5-e41c-4260-a418-b6e5854f3bed-0
00:32:53.440 --> 00:32:56.360
And so now it's time for some
acknowledgments.

d550d6b1-4a12-4d60-8063-7be6704ee008-0
00:32:56.440 --> 00:33:00.451
So I would like to 1st thank the two labs
that I am in,

d550d6b1-4a12-4d60-8063-7be6704ee008-1
00:33:00.451 --> 00:33:02.600
the Haber and Ragunathan labs.

9f3f6d63-06bb-4652-8026-123a625122d1-0
00:33:03.480 --> 00:33:08.825
Jim Haber and Kaushik Gurgunathan are
amazing mentors and I'm so lucky to have

9f3f6d63-06bb-4652-8026-123a625122d1-1
00:33:08.825 --> 00:33:09.840
2P is honestly.

55417f10-1cf3-428c-b2cf-18e6a51d150a-0
00:33:10.800 --> 00:33:15.068
And then I would also like to thank the
community that have build at Brandeis,

55417f10-1cf3-428c-b2cf-18e6a51d150a-1
00:33:15.068 --> 00:33:18.472
especially the friends that I have made
who are actually here,

55417f10-1cf3-428c-b2cf-18e6a51d150a-2
00:33:18.472 --> 00:33:20.039
right here in the front seat.

3092d657-d468-4918-a8ab-3bbd0ff950db-0
00:33:23.000 --> 00:33:25.360
So here is Shell and I in the lab.

84b3cdea-6995-4c8d-bb61-a64e95340b76-0
00:33:25.360 --> 00:33:28.720
And then lastly,
I would like to thank my family.

32d012ba-3ce1-491b-97b1-0ec0c7ed34a0-0
00:33:30.120 --> 00:33:33.560
Only one is here today, my mom,
but my dad is on FaceTime.

49615ce5-f519-49c5-a368-3b9731728756-0
00:33:34.600 --> 00:33:36.240
My little brother is in high school right
now.

15be9b82-bfc6-4b84-a103-0cdd285ad912-0
00:33:38.200 --> 00:33:42.400
But yeah, with that,
I would take any questions after you are.

51925b58-9108-4a61-817c-a44bfa879943-0
00:33:42.480 --> 00:33:46.466
Yeah,
and thank you all for listening as well

51925b58-9108-4a61-817c-a44bfa879943-1
00:33:46.466 --> 00:33:53.400
as someone who has worked with the Haver
lab is someone who goes to Haver, Jim.

dfdeedaf-92d4-4b41-8423-7bebc4cc3c29-0
00:33:53.440 --> 00:33:59.480
He really is tough on students whom he
cares about and he cares a lot, right.

3d8c8dda-1e6f-4be6-8896-206b3e573774-0
00:33:59.480 --> 00:34:03.228
So it is a very fast-paced environment
and you learn a lot,

3d8c8dda-1e6f-4be6-8896-206b3e573774-1
00:34:03.228 --> 00:34:06.040
You do a lot in a very short amount of
time.

e6cdaf91-edf7-420c-9ee6-86856c165737-0
00:34:06.440 --> 00:34:09.160
And you started your PhD straight out of
here.

d9318f62-c758-4ba6-82f6-e3440795de8f-0
00:34:09.160 --> 00:34:13.681
So you had your experience here,
so can you advise students who wanted to

d9318f62-c758-4ba6-82f6-e3440795de8f-1
00:34:13.681 --> 00:34:18.325
pursue a PhD in high school that is
highly competitive and research focused

d9318f62-c758-4ba6-82f6-e3440795de8f-2
00:34:18.325 --> 00:34:22.358
as fertilized university,
what is some advice you have for people

d9318f62-c758-4ba6-82f6-e3440795de8f-3
00:34:22.358 --> 00:34:24.680
that want to pursue a path like yours?

ae715439-eb02-4b87-8086-e5611d15fb65-0
00:34:24.960 --> 00:34:25.280
Yeah.

78c8a3f1-1c50-4b88-930b-695102208082-0
00:34:27.000 --> 00:34:34.798
So I would say what really sort of helped
me the most is seeking out sort of

78c8a3f1-1c50-4b88-930b-695102208082-1
00:34:34.798 --> 00:34:38.040
different kind of opportunities.

cf168bb2-90ad-4ad7-bdb7-ddf88c9f1d46-0
00:34:38.640 --> 00:34:43.185
So the first of course was joining your
lab and getting exposed to to to research

cf168bb2-90ad-4ad7-bdb7-ddf88c9f1d46-1
00:34:43.185 --> 00:34:44.960
in a small way through your lab.

4d1fe29a-bd70-4df2-a182-4be5464973c6-0
00:34:46.080 --> 00:34:49.182
Additionally,
I would also say sort of finding

4d1fe29a-bd70-4df2-a182-4be5464973c6-1
00:34:49.182 --> 00:34:51.360
community within your professors.

d057d169-3b3c-4496-ab1a-7ca4ce3067a8-0
00:34:51.360 --> 00:34:54.731
So as as you know,
all of my professors that are here that I

d057d169-3b3c-4496-ab1a-7ca4ce3067a8-1
00:34:54.731 --> 00:34:57.440
made a community out of sort of helped me
a lot.

be2aca64-5afe-4f85-b8fe-95f020164d23-0
00:34:58.840 --> 00:35:02.394
I would also say that it's,
it may sound scary,

be2aca64-5afe-4f85-b8fe-95f020164d23-1
00:35:02.394 --> 00:35:07.727
however I think that like there's proof
right in front of me right now,

be2aca64-5afe-4f85-b8fe-95f020164d23-2
00:35:07.727 --> 00:35:13.577
which is you will make a community at
whatever sort of school you decide to go

be2aca64-5afe-4f85-b8fe-95f020164d23-3
00:35:13.577 --> 00:35:13.800
to.

83486460-5fb5-4831-8f45-4ebd7d7bbb94-0
00:35:14.840 --> 00:35:16.040
This is proof right over here.

257999c1-107e-4d88-94da-b0ba2f77f7db-0
00:35:16.040 --> 00:35:19.160
They came all the way from Waltham to to
come and watch me talk here.

e62c4e3a-4c3f-4ad6-8401-be2704d68bd2-0
00:35:20.560 --> 00:35:25.751
And additionally, it's, it's not,
it's not just you doing everything on

e62c4e3a-4c3f-4ad6-8401-be2704d68bd2-1
00:35:25.751 --> 00:35:26.400
your own.

526dcb26-b4d9-4d08-a653-3e4565291362-0
00:35:26.920 --> 00:35:30.543
It may sound sort of like, oh, well,
you're the one doing all this work with

526dcb26-b4d9-4d08-a653-3e4565291362-1
00:35:30.543 --> 00:35:32.520
you and your Pi, but you're also in a lab.

4e3b1619-d915-4938-9ec0-839253062c5c-0
00:35:32.640 --> 00:35:36.160
And there's also labs next to you,
above you, below you.

e1628861-e67f-41b2-8792-e383b04ba0c3-0
00:35:36.920 --> 00:35:39.977
And so you're,
you're going to get support through many

e1628861-e67f-41b2-8792-e383b04ba0c3-1
00:35:39.977 --> 00:35:40.960
different avenues.

f36f88d1-ed9a-475d-9c9e-d7f14fad58da-0
00:35:42.320 --> 00:35:45.680
But yeah, I think, I think that's,
that's the advice that that I would give.

27aed9af-e3de-46ab-ac8d-0efa284ef852-0
00:35:46.160 --> 00:35:48.520
Yeah, yes.

f1c58d57-c417-4360-8b9f-dce16ccee394-0
00:35:57.000 --> 00:35:58.120
Truly wonderful call.

829255a8-ae93-424e-86c8-aab2ba285f9e-0
00:35:58.240 --> 00:35:58.480
Thank you.

880fbc20-400a-4e48-be5c-36b0246c0656-0
00:35:59.120 --> 00:35:59.480
Thank you.

e7d9c91f-7460-42ff-b840-b40ee6ebf411-0
00:36:02.000 --> 00:36:08.275
I'm curious about the idea of the
switching is actually an invasion and

e7d9c91f-7460-42ff-b840-b40ee6ebf411-1
00:36:08.275 --> 00:36:12.720
then copying a minute withdrawals, right,
correct.

d7ad2e20-6baf-41db-867c-d8e541dc0e1f-0
00:36:14.000 --> 00:36:16.836
There's lots of questions about how far
in that gather you're trying to figure

d7ad2e20-6baf-41db-867c-d8e541dc0e1f-1
00:36:16.836 --> 00:36:17.160
that out.

30befe0d-9f8d-4281-863f-42d1b6c44fce-0
00:36:18.480 --> 00:36:20.400
But how does that compare?

c7eeff4b-7bd6-4860-af79-e9f3118b6d26-0
00:36:20.400 --> 00:36:25.164
What happens differently when you have
monologous recombination 'cause you're

c7eeff4b-7bd6-4860-af79-e9f3118b6d26-1
00:36:25.164 --> 00:36:29.440
not doing homologous recombination,
copying is not changing anything,

c7eeff4b-7bd6-4860-af79-e9f3118b6d26-2
00:36:29.440 --> 00:36:32.800
but in homologous recombination,
things are exchanged.

acbb4dc3-b557-4802-ab51-41a9873b6744-0
00:36:32.880 --> 00:36:33.120
So what?

96f1321c-50e8-4531-9f2e-1477588fdf97-0
00:36:34.080 --> 00:36:34.920
How does this remain?

068049f9-6ee4-4169-a924-68d9fc692621-0
00:36:35.320 --> 00:36:36.960
Yes, yeah, yeah, yeah, yeah.

d5e0981e-2a35-428d-be7e-b797ccafde8c-0
00:36:37.120 --> 00:36:41.340
So right there,
there are many different sort of kinds of

d5e0981e-2a35-428d-be7e-b797ccafde8c-1
00:36:41.340 --> 00:36:43.160
homologous recombination.

eca626da-2014-4145-abb5-fc7446456782-0
00:36:43.560 --> 00:36:44.160
Yes.

6726dbfc-00d9-47f3-92e5-53a68c764431-0
00:36:45.000 --> 00:36:48.428
So I think the kind that you're thinking
about sort of happens when there's a

6726dbfc-00d9-47f3-92e5-53a68c764431-1
00:36:48.428 --> 00:36:49.440
crossover event, right?

2c5bfa89-a7bc-41ce-b280-4a7adc2fada0-0
00:36:49.440 --> 00:36:54.320
When there's like there's actual like
exchange of DNA sequences, right.

06d0c8e7-4356-4672-a829-44cf3883950c-0
00:36:54.760 --> 00:36:58.410
So this kind of homologous or combination
doesn't have that sort of exchange

06d0c8e7-4356-4672-a829-44cf3883950c-1
00:36:58.410 --> 00:36:59.880
because it is a different kind.

2ee21706-98d8-49b1-a046-eeace9f50a1f-0
00:36:59.880 --> 00:37:01.120
And those do exist.

6f29cb27-f4c0-43b4-80e2-e3e6bd31e7c3-0
00:37:01.160 --> 00:37:01.440
Yeah.

f83d0d86-e45e-44fd-b4c3-3ad1c7470f1a-0
00:37:01.680 --> 00:37:04.797
So there,
there are definitely other contexts in

f83d0d86-e45e-44fd-b4c3-3ad1c7470f1a-1
00:37:04.797 --> 00:37:10.078
which that does occur and which it's not
in meeting type switching obviously since

f83d0d86-e45e-44fd-b4c3-3ad1c7470f1a-2
00:37:10.078 --> 00:37:12.560
nothing happens to that donor template.

25c3f0e0-e24a-406e-b89d-6f95a534d0d4-0
00:37:12.960 --> 00:37:17.037
But there are many other different
contexts in which we have learned that

25c3f0e0-e24a-406e-b89d-6f95a534d0d4-1
00:37:17.037 --> 00:37:18.360
sort of crossover event.

796c7165-176e-45f3-923f-e4e9ca7a02eb-0
00:37:18.760 --> 00:37:22.120
But in this specific situation that
doesn't happen remarkably.

4b5ad617-45f2-4406-9edb-e5fd631cb1ee-0
00:37:22.320 --> 00:37:25.193
It might happen maybe in a small
percentage of cells,

4b5ad617-45f2-4406-9edb-e5fd631cb1ee-1
00:37:25.193 --> 00:37:29.451
but but not enough where we can sort of
detect it through various tools that we

4b5ad617-45f2-4406-9edb-e5fd631cb1ee-2
00:37:29.451 --> 00:37:33.017
use to detect it, You know,
so we can sort of screen different way

4b5ad617-45f2-4406-9edb-e5fd631cb1ee-3
00:37:33.017 --> 00:37:35.040
different outcomes that happen, right.

84d3be7e-9505-424d-a957-54a502ca794d-0
00:37:35.280 --> 00:37:38.720
So this specifically is is called sort of
a gene conversion event.

c9e55211-cae8-4c85-8b38-9486b419a6d8-0
00:37:39.480 --> 00:37:43.080
And we can do a bunch of different things
to sort of analyze that.

f824410a-5df7-4dc7-9713-3375c9238dbb-0
00:37:43.720 --> 00:37:46.520
One simple way is just by doing APCR,
honestly.

2feffb9e-c5f6-4f26-a01f-8b10bfec708d-0
00:37:47.200 --> 00:37:50.806
And so if we were to do that,
we wouldn't be able to detect that small

2feffb9e-c5f6-4f26-a01f-8b10bfec708d-1
00:37:50.806 --> 00:37:53.600
percentage of some cells that maybe have
crossed over.

2a1481c6-50f4-402c-a956-25c37fd925f1-0
00:37:53.840 --> 00:37:59.320
But I it's really unlikely just based on
like past experiments that have been done.

93364a5a-d948-46e3-bf67-ce7697ee0be2-0
00:37:59.320 --> 00:38:02.160
But yeah, that's,
that doesn't occur in this context.

d6d1bdc5-2083-4cae-9336-ef771c76fafe-0
00:38:02.160 --> 00:38:02.720
Right over here.

7c9ccaf3-c557-43e0-848b-623cbb746237-0
00:38:02.760 --> 00:38:03.000
Yeah.

4cf773ec-c33d-40a0-86cc-18a21fc78524-0
00:38:03.000 --> 00:38:12.200
Thank you for a great talk.

20d01116-730b-4bc1-9a17-958579c0a557-0
00:38:12.200 --> 00:38:13.880
And forgive me, I'm a cell biologist.

71c4784d-d514-45cf-a751-48b7fb299ea9-0
00:38:15.120 --> 00:38:21.207
I'm at the organization level, but I am,
but you're talking so good that I'll be

71c4784d-d514-45cf-a751-48b7fb299ea9-1
00:38:21.207 --> 00:38:22.560
able to follow it.

5bc00c54-ca28-420b-825e-66dcf4456e11-0
00:38:22.760 --> 00:38:27.200
And however my question I hope makes
sense.

444bc0b9-32b2-4fb3-af56-7cd5f5fced55-0
00:38:27.640 --> 00:38:32.845
I am just curious as to, you know,
it sounds like some of the yeast can mate

444bc0b9-32b2-4fb3-af56-7cd5f5fced55-1
00:38:32.845 --> 00:38:34.400
switch and some cannot.

6cf805a0-e47d-4581-bfe7-1de4ad40e0e9-0
00:38:34.840 --> 00:38:40.906
And so given what you shared that I was
wondering if the ones that cannot,

6cf805a0-e47d-4581-bfe7-1de4ad40e0e9-1
00:38:40.906 --> 00:38:47.134
are they the ones that have the served
proteins finding to like silence that

6cf805a0-e47d-4581-bfe7-1de4ad40e0e9-2
00:38:47.134 --> 00:38:49.480
area that can't be expressed?

80fd04c5-217a-4a37-bec4-47e57e986bde-0
00:38:51.080 --> 00:38:51.560
Yeah.

2c1cbb46-5c0b-4111-b27c-c163a08813c0-0
00:38:51.720 --> 00:38:54.800
So, so some cells that that can't switch.

866bec72-e857-4f4b-9892-748cba54ee1f-0
00:38:56.680 --> 00:39:02.935
So I think the the ones that that can't
switch probably have some kind of yeah,

866bec72-e857-4f4b-9892-748cba54ee1f-1
00:39:02.935 --> 00:39:07.705
some kind of defect in,
in some kind of small aspect in that

866bec72-e857-4f4b-9892-748cba54ee1f-2
00:39:07.705 --> 00:39:08.800
system itself.

066cd1f9-5327-49c8-a877-97422c26d85b-0
00:39:08.960 --> 00:39:14.182
Whether it be some sort of defect by like
a mutation in in some important protein

066cd1f9-5327-49c8-a877-97422c26d85b-1
00:39:14.182 --> 00:39:17.240
for that heterochromatic donor template
itself.

428564eb-64c1-443a-9cda-31a60d7e3e64-0
00:39:17.440 --> 00:39:22.426
Or it could be something within that
expressed locus itself where perhaps

428564eb-64c1-443a-9cda-31a60d7e3e64-1
00:39:22.426 --> 00:39:26.940
maybe that recognition site is removed in
a way or like, you know,

428564eb-64c1-443a-9cda-31a60d7e3e64-2
00:39:26.940 --> 00:39:30.040
that that HO endonuclease can't cleave
there.

c83c4be9-69cc-4b5e-b3cf-1583a06d9e89-0
00:39:30.040 --> 00:39:32.480
So therefore mating type switching cannot
happen.

bd00adba-c5b2-4813-9b90-1d919e5fa136-0
00:39:32.880 --> 00:39:34.480
It can't be initiated in the 1st place.

95912916-9cad-477e-b01a-958183406024-0
00:39:34.480 --> 00:39:37.254
So, yeah,
I think those are maybe two different

95912916-9cad-477e-b01a-958183406024-1
00:39:37.254 --> 00:39:41.994
areas in that whole process that perhaps
causes some kind of effect in some cells

95912916-9cad-477e-b01a-958183406024-2
00:39:41.994 --> 00:39:43.440
not being able to switch.

f7b6b749-58bf-415c-90a4-37affe6643cc-0
00:39:43.440 --> 00:39:54.560
I'm just curious how much I OK, thank you.

973643ab-2716-444e-8ae9-bf9d96780647-0
00:39:56.680 --> 00:40:03.735
I'm just curious how much is known about
how RAT 51 actually helps in the scanning

973643ab-2716-444e-8ae9-bf9d96780647-1
00:40:03.735 --> 00:40:06.200
to look for Mollus sequences?

8569f302-094f-4628-b733-e7113d5ad54b-0
00:40:07.080 --> 00:40:07.960
How much we know?

df40b7e7-972a-453f-a7e3-5fe7c6e7064f-0
00:40:08.080 --> 00:40:10.440
Yeah, I'm just like, what's the mechanism?

ea108cf4-eb21-4caf-a198-2d25c468a681-0
00:40:12.040 --> 00:40:12.880
Yeah, yeah, yeah.

80350b99-67b0-4492-b2b0-9e2734ce7cc5-0
00:40:12.960 --> 00:40:13.280
That's a.

5578d12d-61f0-4919-8a4b-7e1d247cd20c-0
00:40:13.280 --> 00:40:16.000
That's a loaded question and it's.

9c906a7e-eee6-4b14-b97c-98f896c09259-0
00:40:16.040 --> 00:40:19.660
It's a great question and it is a
question that researchers are still

9c906a7e-eee6-4b14-b97c-98f896c09259-1
00:40:19.660 --> 00:40:21.160
trying to answer to this day.

5728e145-0484-4c00-88fd-c3f48cac8516-0
00:40:21.160 --> 00:40:22.960
It is, it's, it's remarkable, right?

c67da1c3-780f-4afd-b753-6e684643d24c-0
00:40:22.960 --> 00:40:25.444
How can,
how exactly can it find a needle in a

c67da1c3-780f-4afd-b753-6e684643d24c-1
00:40:25.444 --> 00:40:25.920
haystack?

a7e8c9e5-d3fb-41f7-8d1f-7a2dde6206ea-0
00:40:27.360 --> 00:40:30.908
You know, the,
the things that we do know have mostly

a7e8c9e5-d3fb-41f7-8d1f-7a2dde6206ea-1
00:40:30.908 --> 00:40:34.785
come from deleting RAD 51 and then seeing
what happens in,

a7e8c9e5-d3fb-41f7-8d1f-7a2dde6206ea-2
00:40:34.785 --> 00:40:38.400
in several different kind of repair
mechanisms, right?

e3cdf256-564c-4bab-938b-cf959be96113-0
00:40:38.400 --> 00:40:39.680
So I just told you about 1:00.

061cbe71-8ce0-4bcc-8bd8-0fabb8118f62-0
00:40:39.880 --> 00:40:43.120
But that is also important in many of
other different ones.

cb3869c4-bb3f-4559-b882-5732437ce261-0
00:40:43.760 --> 00:40:46.640
And when you delete that protein,
you just, you don't get repair.

f2fb3822-087e-4b2f-9b54-61e19b9601b1-0
00:40:46.640 --> 00:40:48.320
So therefore cells eventually die.

5d04362a-5ab0-4402-a166-4918e8142d67-0
00:40:48.760 --> 00:40:53.245
And so that's how we know that this
protein is is very, very important in,

5d04362a-5ab0-4402-a166-4918e8142d67-1
00:40:53.245 --> 00:40:55.040
in repairing a break at least.

d06fc040-eab1-4ec4-8f78-8e7684d2fe39-0
00:40:55.680 --> 00:40:58.480
But yeah, I like we,
we still don't know so much.

4966f567-e183-41e7-898c-f8ccdc2c7096-0
00:40:58.480 --> 00:41:00.280
It is still still being researched today.

e55a5180-1e84-4f10-97c0-311af7da6996-0
00:41:00.280 --> 00:41:02.388
I,
I asked my Pi that sort of like the first

e55a5180-1e84-4f10-97c0-311af7da6996-1
00:41:02.388 --> 00:41:05.667
time that I was learning all this and
joined his lab and he was like,

e55a5180-1e84-4f10-97c0-311af7da6996-2
00:41:05.667 --> 00:41:07.120
I don't have an answer for you.

90451c40-20f9-484e-b1d6-d001fdae455c-0
00:41:07.120 --> 00:41:10.400
Like this is still things that we are
trying to figure out.

59793252-2932-4219-8287-f018c5c25f7c-0
00:41:10.400 --> 00:41:12.040
Can I just ask a follow up question?

31cbcdee-0cbe-4592-babc-4aa86f40e3a8-0
00:41:12.040 --> 00:41:15.964
Yeah, Are there,
are there they're like mutants that are

31cbcdee-0cbe-4592-babc-4aa86f40e3a8-1
00:41:15.964 --> 00:41:21.196
not null mutants of RAD 51 that like
impair the function that don't totally

31cbcdee-0cbe-4592-babc-4aa86f40e3a8-2
00:41:21.196 --> 00:41:22.159
get rid of it.

84aa9abb-2d3a-4c17-a0cc-a5f69c4ce2c0-0
00:41:22.160 --> 00:41:23.240
That might give some clue.

773fef47-9716-4add-84f5-02dc01fee354-0
00:41:23.480 --> 00:41:24.520
Yeah, absolutely.

2a1a160f-83b6-408d-baea-8c35ee7b029a-0
00:41:24.520 --> 00:41:26.565
Yeah,
there are definitely those kind of

2a1a160f-83b6-408d-baea-8c35ee7b029a-1
00:41:26.565 --> 00:41:28.960
mutants that have been that have been
screened.

27cd4ccc-3c61-442b-8bc7-22222ee73eca-0
00:41:30.040 --> 00:41:33.381
I I can't remember off the top of my head
sort of the different phenotypes that

27cd4ccc-3c61-442b-8bc7-22222ee73eca-1
00:41:33.381 --> 00:41:35.720
like they give you,
but but they're they are out there.

4fc85e53-208d-468b-99d1-6398a8bf1180-0
00:41:35.720 --> 00:41:36.880
Yes, I know that for certain.

2b6880c0-4564-41bf-af99-cde2534efba6-0
00:41:36.880 --> 00:41:37.880
Yeah, yeah.