Lecture 8 - DNA Damage, Tolerance and response

Question 1

What happens if DNA damage is not repaired before replication?

Answer 1

• If mismatch base pair is replicated by the usual replication machinery before being repaired it results in mutation
  
  • Altered/absent bases - DNA lesions
    ○ DNA lesions are not always repaired before replication. Normal polymerases cannot replicate DNA containing damaged bases - leads to stalled replication forks
    ○ DNA damage response is induced in both prokaryotes and eukaryotes
    ○ Specialised trans-lesion synthesis (TLS) DNA pols replicates some DNA with damaged template.

Question 2

What is transleison DNA synthesis?

Answer 2

DNA damage tolerance - trans-lesion DNA synthesis
TLS DNA pols have a more open and flexible active site allowing some replication of damaged DNA but the fidelity of the synthesis is low (don’t put in the correct base/s opposite damaged DNA)
TLS DNA pols lack 3’-5’ proofreading activity meaning they have much higher error rates. They allow continued DNA replication at an unrepaired lesion but with increased risk of incorrect insertion ultimately leading to mutation. However, error prone replication is better than no replication.

TLS synthesis polymerases are recruited to replication forks by interaction with sliding clamp
* The structure of Y-family DNA pol catalytic domains reveal an overall right hand topography however unlike A and B family replicative DNA polymerase they have an additional little finger domain that contacts DNA close to the lesion site
* Because of their low fidelity, TLS polymerases must only be recruited when necessary
○ In bacteria concentrations of TLS polymerase are low but are increased in response to DNA damage

Question 3

What is the DNA damage response

Answer 3

The DNA damage response is induced when there are damaged bases/strand breaks aren’t repaired before replication (dealt with during replication):
* Increase in DNA repair proteins
* Delay in cell cycle
* In multicellular organisms programmed cell death

Question 4

Describe the DNA damage response in bacteria.

Answer 4

Bacteria respond to DNA damage with the SOS response, a mechanism to induce the expression of various proteins including RecA and LexA:
* RecA is a multifunctional DNA binding protein that acts as a damage sensor (also as a recombinase). In the absence of DNA damage RecA is normally inactive
* The LexA protein is a repressor that prevents transcription of many SOS genes by binding to their operators.
LexA binds as a dimer at sequences with similarity to the consensus sequence to block
transcription.
The DNA damage response in bacteria
1. When a replication fork stalls RecA binds to form a filament on ssDNA and becomes activated to cleave LexA repressor
2. Cleaved LexA can’t bind to the operators so SOS genes are transcribed.
Many genes under SOS regulation encode DNA repair proteins:
○ UvrA, B, D proteins - nucleotide excision repair
○ RecA protein, RuvA and RuvB proteins - recombination repair of strand breaks
○ TLS pols - DinB and UmuC
○ SulA protein - cell division inhibitor - increases time window for DNA repair before cell division
3. Induced repair proteins repair DNA damage
4. As DNA is repaired ssDNA decreases, reducing RecA filament assembly, reducing LexA cleavage
5. DinI protein mimics DNA and RecA binds and is sequestered
6. Newly synthesised LexA repressor binds to SOS boxes, SOS genes are repressed.

Question 5

Describe the DNA damage response in eukaryotes.

Answer 5

1. DNA damage sensors recognize damaged DNA
   ○ RPA binds to ssDNA
   ○ MRN or KU bind to double stranded breaks
   
   2. The damage sensors each recruit a different transducer regulatory kinase to the damage site - ATR, ATM, DNA-PKcs
   3. Transducer regulatory kinases activate downstream proteins that, when phosphorylated recruit effector proteins to repair damaged and checkpoint proteins to halt the cell cycle and allow time or repair.

RPA senses single stranded DNA at stalled replication forks
RPA accumulation leads to activation of ATR
Activated ATR phosphorylates targets to modulate multiple aspects of DNA metabolism
Stressed replication forks recruit TLS polymerases

RPA senses single stranded DNA at stalled replication forks
* RPA binds single-stranded DNA at the replication fork (exposed on lagging strand template
* RPA is normally removed as lagging strand template is replicated
* DNA damage causes DNA pol to stall at damage but helicase continues unwinding

RPA accumulation leads to activation of ATR
* RPA recruits ATR via an adaptor, ATRIP - binds to both RPA and ATR
* RPA also recruits a repair specific sliding clamp-clamp loader complex
* The damage specific sliding clamp is known as 9-1-1
* 9-1-1 recruits TOPBP1 and these activate ATR

Activated ATR phosphorylates targets to modulate multiple aspects of DNA metabolism
* Cell cycle control - Checkpoint kinase CHK1
* Replication fork stabilisation - RPA polymerases - slows replication fork progression
* Replication origin control - RPA MCM complex PreRC - Delays replication initiation at origins

Stressed replication forks recruit TLS polymerases
* In addition to recruitment of ATRIP and ATR, RPA bound to ssDNA also recruits a complex that monoubiquitinates PCNA
* Replicative polymerases have reduced affinity for PCNA and are recruited to replication fork
* TLS polymerases have affinity for ubiquitinated PCNA and are recruited to replication fork
TLS polymerases then resume DNA synthesis -error prone, but does at least allow progression of replication fork.

Question 6

Describe DNA double strand break repair.

Answer 6

There are two alternative pathways:
* Non-homologous end joining - simply rejoins the ends - predominant in non-dividing cells
* Homology-directed repair uses homologous DNA as a template. Primarily used in late S phase/g2, when a sister chromatid (identical) is available as template
Non homologous end joining (NHEJ) is the only readily available pathway in G1.
* Nuclease digestion can remove a few nucleotide before ligation
* Resection can expose a simple processing of ends but end processing is usual
This sequence loss means NHEJ is mutagenic

Question 7

How are double strand DNA breaks sensed?

Answer 7

DNA double strand breaks are sensed by the MRN complex
* MRN has Mre11, Rad50 and Nbs1 subunits
* MRN recruits the regulatory kinase ATM
* ATM exists in cells as an inactive dimer
* Interaction of ATM with the MRN/DSB complex leads to autophosphorylation and activation of ATM
ATM phosphorylates targets to modulate multiple aspects of DNA metabolism/cell cycle control
* Recruitment of MRN and ATM
* Signal amplification by recruitment of MDC1 and additional ATM
* Phosphorylation of key effectors - CHK2 /P53

Double strand DNA breaks are sensed by the KU protein
* KU a heterodimer of KU70 and KU80, is a sensor of DNA double stranded breaks
* KU is very abundant and binds strongly to the ends of DSBs
* In vertebrates, KU recruits the regulatory kinase DNA-PKcs to from DNA-PK
* DNA-PK recruits proteins to join broken ends by non-homologous end joining (NHEJ leads to some loss of information at the DSB site).