Chiolo Lecture 3 Flashcards
What does trans-lesion synthesis allow?
damage tolerance, not resistance
What is a disadvantage of trans-lesion synthesis?
highly mutagenic (“error prone”)
What does trans-lesion synthesis replicate with?
replicates across a damage with specific low-fidelity DNA polymerases
What are two pathways DNA damage can go through?
- DNA repair
- DNA damage tolerance > translesion synthesis > DNA repair
What is the process of trans-lesion synthesis in E.coli?
- β-clamp and DNA polymerase III stalls at TT dimer
- a translesion polymerase replaces the β-clamp and DNA polymerase
- translesion polymerase replicates across the TT dimer
- β-clamp and DNA polymerase replaces translesion polymerase with ATP
- replication continues
What is incorporation of bases independent from?
base pairing
How many translesion polymerases are found in humans?
5 translesion polymeraes
What are the two models of trans-lesion synthesis in mammalian cells?
- polymerase-switching at the fork
- gap-filling after the fork
What happens during polymerase-switching?
- happens at the fork
- DNA polymerase stalls at the damaged nucleotide
- PCNA is ubiquitinated
- translesion polymerase replaces β-clamp and DNA polymerase
- translesion polymerase replicates across the TT dimer
- β-clamp and DNA polymerase replaces translesion polymerase with ATP
- replication continues
What triggers the switch to trans-lesion polymerase in mammalian cells?
ubiquitination of PCNA
What happens during gap-filling?
- happens after the fork
- DNA polymerase skips the TT dimer because it is the lagging strand
- translesion polymerases fills the gap via Okazaki fragments
What is an exception polymerase for TT dimers?
in human cells, Polη correctly inserts two As when it encounters a T-T dimer
What is the function of Polη?
T-T dimer fits in the active site of DNA polymerase eta allowing to correctly insert two As across from the dimer
What do Polη mutations result in?
a variant Xeroderma Pigmentosum
What are the symptoms of Xeroderma Pigmentosum?
- UV sensitivity
- skin cancer
What is the difference between individuals with a Polη mutation and a nucleotide excision repair mutation?
individuals live longer, and still have the ability to replicate
What are the sources of double-strand breaks?
- radioactive materials
- cosmic rays
- medical x-ray imaging
- cancer therapy
- nuclear power plants
- scientific research
What does radioactive iodine intake cause?
thyroid cancer
When do double-strand breaks form?
during replication
What are two paths of double-strand breaks?
- a nick that causes fork collapse
- a lesion that causes fork collapse and fork regression
What happens in fork regression?
the replication fork regresses, allowing the synthesized strand with a nick to continue replication and override the lesion
Why are double strand breakages the most dangerous DNA lesions?
a single unrepaired double strand breakage triggers cell death/ genome instability
What errors can DNA replication cause?
Spontaneous Damage
1. AP site (10,000 DSBs/day)
2. base damage (6,000 DSBs/day)
3. DSB (10 DSBs/day)
What does defective repair of double-strand breakages result in?
translocations in cancer cells
What context does double-strand breakage occur in?
chromatin context
What types of modifications can occur on histone tails?
- methylation
- acetylation
- phosphorylation
- ubiquitylation
What are the four types of histones?
- H2A
- H2B
- H3
- H4
What induces cell cycle arrest and DSB repair?
check-point mediated phosphorylation of H2A variants
What is the function of H2A/H2Ax?
phosphorylation promotes DSB repair by recruiting chromatin remodelers
What is the histone variant for yeast?
H2A
What is the histone variant for humans?
H2Ax
What is the histone variant for flies?
H2Av
What are two chromatin remodelers?
Swr1 and Ino80
What is the function of Swr1 and Ino80?
remodel chromatin by sliding nucleosomes along the DNA or exchanging histones within nucleosomes. This allows for more protein recruitment
What is Tip60?
a histone modifier (acetyltransferase) that causes DNA to be more neutral and dissociate less with histones
What do mutations in ATM or ATR lead to?
Ataxia Telangiectasia (A-T)
What are the symptoms of Ataxia Telangiectasia?
- progressive difficulty with coordinating movements
- enlarged blood vessels telangiectases, vascular lesions
- cancer predisposition (leukemia), sensitivity to radiotherapy and medical X-rays
- immune defects
- sterelity
What are direct consequences of defective DSB repair?
- cancer predisposition (leukemia), sensitivity to radiotherapy and medical X-rays
- immune defects
- sterility
What is the process of DSB repair?
- a double strand breakage occurs
- ATM and ATR recognizes the breakage and phosphorylates each nucleosome
- chromatin remodelers, SWR1 and Ino80, bind to the nucleosomes, dissociating each histone
- removal of nucleosomes promotes protein recruitment for repair, MRX, MEC1
