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L05v04
[00:00:00.00]
[00:00:02.18] PROFESSOR: Hi. In this video, we'll talk about three different ways that cells
repair damaged DNA-- base excision repair, nucleotide excision repair, and double-strand break
repair.
[00:00:15.67] Method 1-- base excision repair. This occurs when a single base is affected. In the
case shown here, it is a type of damage called deanimation. This is when a C base has an amino
group that gets deaminated, leaving a carbonyl group in its place. And coincidentally, when this
happens, the result is the base of cytosine turns into a base of uracil-- the base that is normally in
RNA.
[00:00:42.14] Now you will remember that uracil acts the same and base pairs the same as a T.
And so this seems, to DNA, like a C-to-T mutation. But the cell also knows that there should be
no uracil in DNA.
[00:00:55.54] In fact, there are special enzymes called uracil DNA glycosylases that will scan the
genome for places where a U is present. When it finds it, it will excise it, cut out the base, and
then cut out the corresponding backbone groups-- a sugar and a phosphate group. Then DNA
polymerase will come along and insert the correct base-- a C, to base pair with the G. And then
the enzyme DNA ligase will seal the nick.
[00:01:21.18] There's a very similar type of DNA damage called depurination, which happens to
purines, which are Gs or As. This class of mutations cleave the bond between the sugar and the
base, and the bases will be lost. So those types of mutations or DNA errors will use this repair
mechanism as well.
[00:01:40.59] These are both two common kinds of mutations, and they are repaired effectively
by the cell, but not perfectly. Historically, we can actually see that, evolutionarily, there are more
C-to-T mutations than other types of base-pair changes. So again, it's a very commonly occurring
event. And the cell catches the vast majority, but not all of them.
[00:02:02.92] A second type of DNA repair method is called nucleotide excision repair. In this
case, two or more bases can be affected. And the type of damage shown here is a pyrimidine
dimer, which is a more generic term for the type of damage we saw in the previous video,
thymine dimers.
[00:02:19.84] So this is a pyrimidine dimer. Instead of a T and T, a thymine dimer, we have a CT base pair, which can also form this dimer. And once again, there are proteins which constantly
scan and surveil the genome. When it finds this kind of an error, it will cleave the backbone at
some distance from the lesion site, remove the entire piece of DNA using the enzyme DNA
helicase, and DNA polymerase will fill in the template, from three prime to five prime, and the
ligase will seal the nick-- final nick. So again, this is an efficient mechanism for repairing
thymine dimers.
[00:02:55.26] Before we get to the third mechanism of repair, we're going to ask the question-when the cell encounters a mutation drawn mostly as a bubble, how does it know which strand to
repair? This picture is not very realistic, because, really, the bubble is on both sides. This picture
implies that the correct base is straight and has a normal backbone, and the incorrect base
bubbles out a bit. But in reality, both sides will be affected.
[00:03:23.15] In this case, you have a C-to-A mutation. After replication, a T will be inserted
next to the A, and a C will properly be inserted next to the G. And going on, you would have
50% mutated cells and 50% nonmutated cells.
[00:03:40.56] Now let's look at what happens if the cell knows somehow that the A is the
incorrect base-- although we don't know how it does that, yet. It can correct the A and then it can
put two G's there. And now you'll have 100% correct cells of the progeny, which will all have
the correct genome.
[00:03:59.66] But think about the consequences if the incorrect base is repaired-- if the G is cut
out, and it gets converted to a T. Now you have 100% of the progeny cells having the mutation
incorporated.
[00:04:10.53] So how does the cell figure out which is the proper base to go at that position? The
answer is quite ingenious. And what the cell does-- which gets it right most of the time, but not
every time-- is it will, once it finds a mismatch, it will scan along the genome in both directions,
looking for the closest nick in the backbone of the strand.
[00:04:32.44] The cell then assumes that this is the most recently synthesized strand, the other
strand, with no nicks, having stood the test of time, per se. And so it will decide to cut out the
mutated region of the DNA that's on the strand that has the closest preexisting nick. And it will
repair that with a polymerase and then a ligase sealing the final nick.
[00:04:54.27] As you might guess, this all happens relatively quickly, right after DNA synthesis.
It has to be quick because if a different enzyme happens to notice there's a nick present in the
DNA it will repair the nick. Now the cell will not know which is the newly synthesized strand.
So after synthesis, the DNA is basically a ticking time bomb, where the cell has to repair this
bulge before another enzyme fixes this remote nick, if it's going to use this mechanism for
pricking out which strand to repair.
[00:05:26.99] Now the third DNA repair mechanism that we'll study is a mechanism called
homologous recombination. And this is a way that cells repair, most, of the time, the most
serious of DNA lesions-- double-strand breaks. When both strands are mutated or damaged, the
cell needs to repair both strands, not just one.
[00:05:46.34] One crude, brute-force method, shown here on the left, is nonhomologous
enjoining. When a double-strand break is identified, if it cannot use homologous recombination,
the cell will just cut back the ends of the DNA to make them blunt, or even, and then just jam the
two ends together. You have deleted DNA base pairs, in this method, which, for a protein, could
be quite disastrous. Losing even a single base will change the reading frame, which can then
change every subsequent amino acid. And while that's better than having a broken chromosome,
nonhomologous enjoining is a last resort.
[00:06:28.52] A preferred method which we'll talk about is homologous recombination. And
what's important to remember is that you have two copies of every chromosome. You have one
chromosome from your father and one chromosome from your mother.
[00:06:40.51] And what the cell will do is use the other chromosome to guide the repair. It will
cut away the affected region. It will use, as a template, the other chromosome. And it will
synthesize new DNA. So it will change your genetic material, replacing, for instance, your copy
of your dad's gene with your mother's version.
[00:06:59.69] So now you will have two copies of the maternal version. But hopefully it won't
incorporate a deleterious mutation. And this is a far preferable alternative to simply jamming the
two ends back together.
[00:07:14.93] This is the molecular mechanism for homologous recombination. In this example,
we have a double-strand break. First, the cell uses an exonuclease to chew back from the breaks
in the five-prime direction. The purpose is to create a segment of the DNA which is singlestranded and then available for base pairing.
[00:07:34.93] Miraculously, the single-stranded session aligns with the other parental
chromosome and now is part of a double helix there. This is called a D-loop. And it can go on
synthesizing in the normal five prime to three prime direction, where the bulge of the other
strand of the paternal chromosome is being successively displaced, until it has traveled some
distance.
[00:07:56.38] Now it has enough material to reach the existing maternal chromosome. And then
the ligase can seal the nick. At the same time, the existing part of the strand can now be used as a
template for the synthesis of the other strand. And again, the ligase will seal the nick here. So
overall, this is pretty amazing.
[00:08:17.61] One of the ways that double-strand breaks occur is through ionizing radiation, like
x-rays or cosmic rays. But a lot of us don't get exposed to x-rays that often. But a common way
that double-strand breaks form is when a replication fork encounters a nick in the backbone.
[00:08:35.32] If you have a leading-strand synthesis and you encounter a nick, that's it. You're
done. There's no further extending, because you no longer have a complete strand that is in this
proximity as a template. The cell will begin homologous recombination by using the exonuclease
activity in the five-prime direction and having a strand that can base pair to the other
chromosome and start the repair process.
[00:09:01.63] We will encounter homologous recombination later in the course, when we're
studying meiosis. As you already know, you inherit one chromosome from your father and one
chromosome from your mother. But the chromosomes that you will pass on to your offspring are
not purely either your paternal or your maternal chromosome, but a combination chromosome
that is unique to you.
[00:09:20.04] You might be wondering, how is it that, in some cases, homologous recombination
can exchange genetic material, versus sometimes simply repairing a damaged segment? So we'll
spend just a few minutes dissecting the mechanism.
[00:09:35.91] The key to this is to focus on this particular intermediate called a Holliday
junction. That's a rather famous term, and the difference is how these crossover intermediates get
resolved. If we cleave the chiasma or cross over this way, you will have an exchange of genetic
material. If you cleave this intermediate that way, you will have a repaired chromosome.
[00:09:59.73] It's impossible to fully visualize here, but let me try to make it a little clearer. On
the left is the form that was shown in the previous slide. On the right is the same molecule, but
it's unwound in this direction. And now we have an open form. And I think you can see here, if
you cleave it this way, that in one case you'll have repaired material, or in this case, if you cleave
it here, you'll have translocation, with some of the paternal and maternal material mixed.
[00:10:28.37] And this is made even clearer here, on this slide. Cut it vertically, and you have
double-strand break repair. Cut it horizontally, and you have crossing over, as in meiosis. OK.
This is a bit challenging to visualize, but work through it, and I think you'll be able to see the
molecular difference.
[00:10:44.76] So that concludes the video on three different types of DNA repair-- base excision
repair, nucleotide excision repair, and homologous recombination. Thanks for listening.