20. DNA repair

Last updated on November 19, 2018 at 17:16

Summary

  • There are multiple types of DNA damage, and multiple types of DNA repair to fix them
  • Nucleotide-excision repair fixes structural changes like pyrimidine dimers. This is done by ABC exinuclease, helicase, DNA pol I and DNA ligase
  • Base-excision repair fixes damaged bases. This is done by DNA glycosylase, AP endonuclease, DNA pol I and DNA ligase.
  • Direct repair fixes methylation and pyrimidine dimers. This is done by DNA photolyases, O6-methylguanine-DNA methyltransferase, and AlkB proteins.
  • Mismatch repair repairs DNA damage caused by when DNA polymerase has inserted a base that’s not corresponding to the base on the other strand (like pairing a G with an A). This is repaired by Mut protein complexes, helicase, exonuclease and DNA polymerase III

Nucleotide-excision repair

While exonucleases remove can remove nucleotides on the end of a DNA-strand, they can’t remove nucleotides in the middle of one. Endonucleases can (endo vs exo, in vs out). However, to remove a fragment of DNA, many nucleotides at a time, an excinuclease is needed.

When DNA damage like pyrimidine dimers are detected, we need ABC excinuclease to remove the damaged fragment. DNA helicase is also needed, as seen on the figure. Then DNA polymerase I or ε fills the empty space, and DNA ligase implements the newly synthetized DNA fragment into the strand.

How nucleotide-excision repair works. The left pathway is for bacteria, the right for humans. You might notice that humans don’t use polymerase I -> III, but ε and other greek letters instead. Also, we use the name ABC excinuclease instead of human excinuclease.

Base-excision repair

This type of repair functions similarly to nucleotide-excision repair. We have a case of a damaged base which DNA glycosylate removes. Now that we have removed the base (but not the whole nucleotide), we are left with an apurinic or apyrimidic residue (a nucleotide without its purine or pyrimidine base). AP endonuclease creates a nick, which DNA pol I fills in, and DNA ligase fixes.

How base-excision repair works.

Methyl-directed mismatch repair

Let’s say that DNA polymerase replicated DNA, but made one mistake. The new strand is error-free, except at one place, where there is now a C where there should have been a T. There is now a mismatch, because the original strand had an A at that spot (so the newly synthesized one should have a T), so the C is obviously misplaced. This obviously has to be fixed, but how does the reparation machinery know which strand is the original, correct one, and which is the newly synthesized one with the error?

All DNA is methylated to some degree. Many bases along the DNA molecule has extra methyl-groups attached to them. After DNA replication, the new strand needs to be methylated in the same way as the original is. However, this methylation does not occur immediately after DNA replication – so the newly synthesized strand isn’t methylated right after it is synthesized. This means that the repair machinery has a small window of time (a few minutes), where it knows which strand is the original one (the one that is methylated), so it can fix the other one.

On the figure on the right, you can see the mismatch in the middle of the strands. The blue strand is the original (because it’s methylated), and the red is the new one, which has to be fixed. The Mut-complex binds to both strands at the site of the mismatch, and then goes along both strands in each direction, making a loop in the DNA.

When the Mut-complex reaches a methyl-group, it knows which strand is the original, and makes a cut on the non-methylated strand, at the site where the other strand is methylated. Now that there is a nick in the new strand, exonucleases can remove every nucleotide from the nick, back to the mismatch, which DNA polymerase III can fill in.

After every mismatch like this has been fixed, Dam methylase will methylate the new strand to be equal to the original one, and the two strands are now undistinguishable. There’s no way to know which one was the original now.

Methyl-directed mismatch repair

Direct repair

How methylation will cause mutations

As discussed in the previous chapter, bases can be methylated by certain chemicals called alkylating agents. When bases are methylated, they can turn into other bases, which will cause a loss of information in the DNA.

Let’s say there is a guanine in the DNA strand. On the other DNA strand, a cytosine is bound to this guanine. If this guanine is methylated, it will become an O6-methylguanine. This base pairs with thymine, and not cytosine, so a mismatch has now occurred, but it cannot be fixed, because both strands are methylated. The mismatch will stay until the DNA is replicated. When it is, the two strands will be separated, so one strand will have the O6-methylguanine while the other has cytosine. When the first strand is replicated, the new strand will contain a thymine, while when the second strand is replicated, its new strand will contain a guanine. Now we have two different DNA molecules, which is bad.

This is repaired by direct repair, which means that the bases are fixed, and not removed and replaced.

Direct repair of O6-methylguanine is done by O6-Methylguanine-DNA methyltransferase. This enzyme isn’t strictly an enzyme, because it can only be used just once. The methyl group from O6-methylguanine is transferred onto the “enzyme” itself, which is now permanently inactive.

Another enzyme that can fix base methylation is AlkB. It needs α-ketoglutarate and Fe2+ to work.

Pyrimidine dimers can be fixed by DNA photolyase. This needs FADH2. It doesn’t occur in humans, so I don’t think it’s important.

What happens if DNA damage happens at both strands at the same time? Then there’s no template to regenerate DNA from. This type of damage is fatal to the cell in most cases, but a protein called BRCA1 will try to fix the damage by inserting random nucleotides. If it works, great, the cell survives, but if it doesn’t (most likely), the cell dies, which it would have anyway.

Help, the damage is too great! How do I kill myself?

Sometimes, damage to the DNA is so severe that the cell better kill itself to not become cancerous. When DNA damage is detected, an enzyme called PARP will begin synthesizing a polymer. This polymer is what signals the DNA-repairing enzymes to start working. However, this enzyme consumes NAD+ when it created this polymer. If the DNA damage is too great, this enzyme will consume all of the cells NAD+ trying to create this polymer. If a cells pool of NAD+ is depleted, it dies. This is how cells commit apoptosis when the damage to the DNA is too great.


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19. DNA replication

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21. RNA metabolism

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