DNA Repair and Genome Integrity

Lecture 8

What are the possible changes in DNA?(depurinations)

cytosine deamination (amine group removed)

Guanine oxidation

methylation (C added)

What is the problem that base modifications can cause?

they can block the activity of DNA polymerases used during DNA replication

What are mutations?

permanent, transmissible changes to the genetic material of a cell

What are Mutagens?

Chemical factors that increase the frequency of mutations (things that cause mutations)

What do DNA polymerases have that help increase fidelity?

the DNA polymerases (pol delta and epsilon) alone introduce errors, however they also have proof reading exonuclease activity which helps reduce errors

What are the implications of mutation rates in health associated with fathers?

Since germs cells are continuously dividing, as men age, mistakes increase and it makes it more likely to pass down a genetic mutation to offspring 

What are the implications of mutation rates in health associated with viruses?

viruses have even less checkpoints than humans which can cause mistakes to go undetected but it also contributes to their evolution

If a mutation is incorporated into a persons DNA it can change the genome

Which DNA polymerase does not have exonuclease/proofreading activity?

Pol alpha because it doesn’t add very many base pairs 

How does mismatch repair work?

acts in proximity to the replisome

once strands are synthesized it gets put onto the strands and fixes the base pairings 

how does mismatch repair find the mismatch and how does it fix it?

it looks for the new strand that has just been synthesized

MSH2 and MSH6 bind to the daughter strand once mismatch is recognized 

this triggers MLH1 endonuclease and PMS2 to make cuts near the mismatch

DNA helicase unwinds while DNA exonuclease digests the segment of the daughter strand with the mismatch 

the gap is repaired by pol delta (lagging strand polymerase) and DNA ligase

how does base excision repair identify the mistake?

looks for repairs where the bases have been chemically modified (like deamination)

EXAMPLE: Deamination - when cytosine gets chemically modified becoming uracil which is now paired with guanine 

if base excision repairs works it will get fixed and change back to a C

IF IT DOES NOT get fixed you get mutant DNA when it is replicated where U will match with A

how does base excision repair fix the problem?

DNA glycosylase hydrolyzes the bond between the mispaired base and the sugar phosphate backbone - removes on the base (in this case its UNG)

then APE1 endonuclease cuts the DNA backbone

AP lyase removes the backbone where there is no base and recruits DNA pol beta and ligase which then insert and ligate the new base to the rest of the strand 

How does nucleotide excision repair identify the problem?

looks for big lesions/changes in the DNA helix

EXAMPLE: the formation of thymine-thymine dimers

ultraviolet light creates bonds between two T causing a change in structure

how does nucleotide excision repair work?

complex protein 23B and XP-C recognize the lesion

they then recruit the transcription factor TFIIH which catalyzed the unwinding of DNA region around the lesion

that then recruits RPA and XP-G which also has unwinding activity

finally XP-F is recruited as well as XP-G and they both have endonuclease activity generating cuts 24-32 base pairs apart releasing the region with the lesion which gets degraded

• XP-F cuts at 3’ end

• XP-G cuts at 5’ end

the gap is then filled by DNA polymerase and joined together by DNA ligase

What is translesion synthesis?

if you have tried everything else and nothing worked this is the last resort

its used just to be able to finish replication - it doesn't actually fix anything

DNA must be in double stranded form

use a special type of DNA polymerase called translesion TLS polymerases - able to bypass the lesion using damaged DNA as the template

they often insert the incorrect base and do not have proofreading exonuclease activity