DNA damage and repair/tolerance/response

Question 1

What range does UV light fall on the spectrum?

Answer 1

100-400nm

Question 2

What does UV radiation do?

Answer 2

promote the formation of intrastrand cross-linked pyrimidines

Question 3

Ames test

Answer 3

assays the potential mutagenicity of chemicals
1. histidine autotroph (bacteria)
2. separate samples into two agar plates (one normal one defined minimal)
3. add mutagen to both + incubate for 12hrs
4. not mutagen = if plates similar
mutagen = more colonies on m.m.

Question 4

How do cells repair DNA damage?

Answer 4

1. excision of damaged DNA (mismatch repair, base excision repair, nucleotide excision repair)
2. direct reversal of DNA damage (enzymatic photoreactivation, repair of O^6-alkylguanine)

Question 5

Mismatch repair (7+1)

Answer 5

1. MutS locates mismatch site
2. MutS recruits and forms MutSLH complex with MutL and MutH
3. MutH identifies new strand (not methylated)
4. MutSLH complex bunches up the DNA
5. MutH activated and ‘nicks’ the new strand at the 5’ end of the MM
6. UvrD (helicase) unwinds the strands and an exonuclease VII degrades it (5’ –>3’)
7. DNA is resynthesises by DNA pol
   ~ this process can also detect + repair hairpins

Question 6

Base excision repair (3+1)

Answer 6

1. DNA glycosylase recognises damed base, flips and exposes it before removing it
2. AP endonuclease recognises abasic site and breaks its surrounding phosphodiester bonds (backbone)
3. DNA pol + ligase add correct nucleotide
   ~ different glycosylases for diff types of damage

Question 7

Nucleotide excision repair (1+5+1)

Answer 7

repairs bulky legions that distort the DNA helix
1. UvrA + UvrB scan DNA for distortion
2. UvrB separates the strands of the damaged region + released UvrA
3. UvrC ‘nicks’ damaged DNA
4. UvrD unwinds the damages DNA + releases UvrC
5. DNA is resynthesised by DNA pol 1
~ protein’s genes under SOS control

Question 8

Enzymatic photoreactivation

Answer 8

1. UV light causes pyrimidine dimers
2. DNA photolyase binds to dimer
3. light hits the enzyme + bond is broken
   OR
4. e- from light photon is transferred to FADH = FADH-
5. e- transfer from FADH- to dimer, causing it to split, e- then returns to FADH

Question 9

Repair of O^6-alkylguanine

Answer 9

~ methyl transferases remove base methylation
~ alkyl transferases repair alkylation of guanine or backbone
E.G. E.coli - Ada
1. Alykl group transferred to Ada
2. Methyl-Ada increases its own expression and expression of ALKA glycosylase
3. ALKA glycosylase removes methylated guanine bases in BER

Question 10

RecA

Answer 10

multifunctional DNA binding protein that acts as a damage sensor + recombinase

Question 11

LexA

Answer 11

repressor protein tha prevents transcription of many SOS genes by binding to their operators
~ binds as a dimer

Question 12

Homology-directed repair

Answer 12

uses homologous DNA as a template to repair DNA

~ used in late S/G2 phase when a sister chromatid is available (template)

Question 13

Non homologous end joining

Answer 13

simply rejoins the ends of a double strand break

~ predominant in non-dividing cell as no sister chromatid (template)

Question 14

Sensor proteins

Answer 14

detect long stretches of ssDNA or dsDNA breaks

~ recruit regulator kinases (ATM or ATR) to mediate damage response

Question 15

mediators and effectors

Answer 15

when phosphorylated by kinases lead to changes in transcription and activate cell-cycle checkpoints

Question 16

MRN complex

Answer 16

~ contains: Mre11, Rad50, Nbs1 (Xrs2 in yeast)

~ interacts with dsDNA breaks and holds ends together

Question 17

What are the three areas that activation of ATR affects?

Answer 17

cell cycle control (arrests it)
replication fork stabilisation (slows its progression)
replication origin control (delays initiation)

Question 18

What are the three areas that activation of ATM affects?

Answer 18

cell cycle control (arrests it)
chromatin modification (provide landing site for repair proteins)
recruitment of repair proteins (DSB or NHEJ)

Question 19

RPA

Answer 19

replication protein A
~ binds ssDNA + builds up if the fork stalls
~ recruits ATR via interaction with the ATR-interating protein
~ recruits TLS polymerase by monoubiquitinating PCNA = pol D/E low affinity but TLS has high affinity

Question 20

TLS DNA Polymerase

Answer 20

~ more open and flexible active site
~ low fidelity
~ no 3’–>5’ proofreading ability - error-prone rep is better than no rep
~ right hand topology but with extra little finger domain
~ under SOS control

Question 21

SulA

Answer 21

~ cell division inhibitor that increases repair time window

~ under SOS control

Question 22

Din1 protein

Answer 22

~ DNA mimic that RecA binds to

~ under SOS control