Chiolo Lecture 3

Question 1

What does trans-lesion synthesis allow?

Answer 1

damage tolerance, not resistance

Question 2

What is a disadvantage of trans-lesion synthesis?

Answer 2

highly mutagenic (“error prone”)

Question 2

What does trans-lesion synthesis replicate with?

Answer 2

replicates across a damage with specific low-fidelity DNA polymerases

Question 2

What are two pathways DNA damage can go through?

Answer 2

1. DNA repair
2. DNA damage tolerance > translesion synthesis > DNA repair

Question 2

What is the process of trans-lesion synthesis in E.coli?

Answer 2

1. β-clamp and DNA polymerase III stalls at TT dimer
2. a translesion polymerase replaces the β-clamp and DNA polymerase
3. translesion polymerase replicates across the TT dimer
4. β-clamp and DNA polymerase replaces translesion polymerase with ATP
5. replication continues

Question 2

What is incorporation of bases independent from?

Answer 2

base pairing

Question 2

How many translesion polymerases are found in humans?

Answer 2

5 translesion polymeraes

Question 2

What are the two models of trans-lesion synthesis in mammalian cells?

Answer 2

1. polymerase-switching at the fork
2. gap-filling after the fork

Question 3

What happens during polymerase-switching?

Answer 3

1. happens at the fork
2. DNA polymerase stalls at the damaged nucleotide
3. PCNA is ubiquitinated
4. translesion polymerase replaces β-clamp and DNA polymerase
5. translesion polymerase replicates across the TT dimer
6. β-clamp and DNA polymerase replaces translesion polymerase with ATP
7. replication continues

Question 3

What triggers the switch to trans-lesion polymerase in mammalian cells?

Answer 3

ubiquitination of PCNA

Question 3

What happens during gap-filling?

Answer 3

1. happens after the fork
2. DNA polymerase skips the TT dimer because it is the lagging strand
3. translesion polymerases fills the gap via Okazaki fragments

Question 3

What is an exception polymerase for TT dimers?

Answer 3

in human cells, Polη correctly inserts two As when it encounters a T-T dimer

Question 3

What is the function of Polη?

Answer 3

T-T dimer fits in the active site of DNA polymerase eta allowing to correctly insert two As across from the dimer

Question 4

What do Polη mutations result in?

Answer 4

a variant Xeroderma Pigmentosum

Question 5

What are the symptoms of Xeroderma Pigmentosum?

Answer 5

1. UV sensitivity
2. skin cancer

Question 6

What is the difference between individuals with a Polη mutation and a nucleotide excision repair mutation?

Answer 6

individuals live longer, and still have the ability to replicate

Question 7

What are the sources of double-strand breaks?

Answer 7

1. radioactive materials
2. cosmic rays
3. medical x-ray imaging
4. cancer therapy
5. nuclear power plants
6. scientific research

Question 8

What does radioactive iodine intake cause?

Answer 8

thyroid cancer

Question 9

When do double-strand breaks form?

Answer 9

during replication

Question 10

What are two paths of double-strand breaks?

Answer 10

1. a nick that causes fork collapse
2. a lesion that causes fork collapse and fork regression

Question 11

What happens in fork regression?

Answer 11

the replication fork regresses, allowing the synthesized strand with a nick to continue replication and override the lesion

Question 12

Why are double strand breakages the most dangerous DNA lesions?

Answer 12

a single unrepaired double strand breakage triggers cell death/ genome instability

Question 13

What errors can DNA replication cause?

Answer 13

Spontaneous Damage
1. AP site (10,000 DSBs/day)
2. base damage (6,000 DSBs/day)
3. DSB (10 DSBs/day)

Question 14

What does defective repair of double-strand breakages result in?

Answer 14

translocations in cancer cells

Question 15

What context does double-strand breakage occur in?

Answer 15

chromatin context

Question 16

What types of modifications can occur on histone tails?

Answer 16

1. methylation
2. acetylation
3. phosphorylation
4. ubiquitylation

Question 17

What are the four types of histones?

Answer 17

1. H2A
2. H2B
3. H3
4. H4

Question 18

What induces cell cycle arrest and DSB repair?

Answer 18

check-point mediated phosphorylation of H2A variants

Question 19

What is the function of H2A/H2Ax?

Answer 19

phosphorylation promotes DSB repair by recruiting chromatin remodelers

Question 20

What is the histone variant for yeast?

Answer 20

H2A

Question 21

What is the histone variant for humans?

Answer 21

H2Ax

Question 22

What is the histone variant for flies?

Answer 22

H2Av

Question 23

What are two chromatin remodelers?

Answer 23

Swr1 and Ino80

Question 24

What is the function of Swr1 and Ino80?

Answer 24

remodel chromatin by sliding nucleosomes along the DNA or exchanging histones within nucleosomes. This allows for more protein recruitment

Question 25

What is Tip60?

Answer 25

a histone modifier (acetyltransferase) that causes DNA to be more neutral and dissociate less with histones

Question 26

What do mutations in ATM or ATR lead to?

Answer 26

Ataxia Telangiectasia (A-T)

Question 27

What are the symptoms of Ataxia Telangiectasia?

Answer 27

1. progressive difficulty with coordinating movements
2. enlarged blood vessels telangiectases, vascular lesions
3. cancer predisposition (leukemia), sensitivity to radiotherapy and medical X-rays
4. immune defects
5. sterelity

Question 28

What are direct consequences of defective DSB repair?

Answer 28

1. cancer predisposition (leukemia), sensitivity to radiotherapy and medical X-rays
2. immune defects
3. sterility

Question 29

What is the process of DSB repair?

Answer 29

1. a double strand breakage occurs
2. ATM and ATR recognizes the breakage and phosphorylates each nucleosome
3. chromatin remodelers, SWR1 and Ino80, bind to the nucleosomes, dissociating each histone
4. removal of nucleosomes promotes protein recruitment for repair, MRX, MEC1