L8: DNA damage tolerance and response Flashcards
How would they be repaired?…
- Mismatched base pairs
- Altered/absent bases
- Replicated by usual replication machinery, resulting in mutation
- Cannot be replicated normally; specific DNA pols can replicate, tolerating damage
What happens when normal replicative polymerases (III, delta, epsilon) try to replicate damaged bases (give examples of base damage)
- Replication fork will stall
(e.g. CPDs, 8-oxoG) - DNA damage response induced (Prok. + Euk)
- Specialised translesion synthesis (TLS) DNA pols replicate some DNA w/ damaged template
TLS DNA pols in E.coli and in humans (categorised by family)
E.coli
- Y-family: DNA pol IV, DNA pol V
Human
- Y-family: Pol eta, Pol iota, Pol kappa
- B-family: Pol zeta
How do TLS pols differ from normal DNA pols? (and what is the consequence?)
- More open, flexible active site (allows some replication of damaged DNA)
-> low fidelity synthesis - Specialised ‘little finger’ domain
- Lack 3’-5’ proofreading activity
-> error rate 10 -2 -10 -4 - At an unrepaired lesion, DNA synthesis may continue but w/ a increased risk of incorrect insertion (leading to mutation)
Interaction between TLS pols and sliding clamp
- Y-family ‘little finger’ domain interacts with beta clamp or PCNA (depending on organism).
- Domain contacts DNA close to lesion site
Levels of TLS pols
- Generally must remain low (due to their inaccuracy); must only be recruited when necessary
- In bacteria, concentrations of TLS polymerases are low but increase in response to DNA damage
What two scenarios lead to induction of DNA damage response?
- Large regions of ssDNA (as a result of Pols encountering lesions -> stalling)
- DNA DSBs (as a result of ss nicks)-> particularly dangerous for cells as the DNA ends can promote lethal Chr. rearrangements
Inducing DNA damage response (first step + consequences)
- ssDNA and DNA DSBs are recognised by damage sensor proteins
- Increased DNA repair proteins
- Delayed cell cycle
- Programmed cell death (only in multicellular organisms)
Key DNA damage response proteins (when inactive - bacteria)
SOS response (>40 proteins induced)
- RecA: multifunctional DNA binding protein, acts as a damage sensor (and recombinase). Normally inactive
- LexA: repressor that prevents transcription of many SOS genes by binding as a dimer to their operators (at sequences w/ similarity to consensus sequence for blocking transcription)
Key DNA damage response proteins (when active - bacteria)
SOS response
- RecA: Binds to ssDNA when replication fork stalls, forms a filament, becomes activated to cleave LexA repressor
- Cleaved and inactivated LexA can’t bind DNA, SOS genes transcribed
Examples of genes under SOS regulation by LexA - bacteria
- DinI
- (NER) UvrA, B, D
- (Recomb. repair) RecA, RuvA, RuvB
- (TLS pols) pol IV, polV
- SulA (inhibits cell division -> greater time window for repair)
Re-establishing repression of SOS genes after repair (3 drivers) - bacteria
- Induced repair proteins repair DNA damage
- As DNA is repaired ssDNA decreases, reducing RecA filament assembly, reducing LexA cleavage
- DinI protein (DNA mimic - acidic residues resemble backbone) is bound by RecA -> RecA sequestered
- Newly synthesised LexA repressor binds to SOS boxes, SOS genes are repressed
DNA damage response overview -Eukaryotes
- DNA damage sensors: RPA (binds ssDNA), KU (binds DSBs)
- Each recruit different transducer regulatory kinase to damage site (ATR, ATM, DNA-PKCS)
- These activate downstream proteins, when phosphorylated, recruit effector proteins (repair damage) and checkpoint proteins (halt cell cycle)
RPA - Eukaroytes
- Senses ssDNA at stalled replication forks (exposed on lagging strand template)
- Remains bound instead of being removed in process of replication when DNA pol stalls
- Recruits ATR via ATRIP (binds to both)
- Also recruits repair-specific SC loader-complex (9-1-1; consists of RAD9-RAD1-HUS1)
- This recruits TOPBP1, activates ATR
ATR activity (key processes w/ examples of targets)
- Cell cycle control
e.g. CHK1, arrests cell cycle -> time window - Replication fork stabilisation
e.g. RPA, Pols, Rad17-Rfc, 9-1-1; slow replication fork progress - Replication origin control
e.g. RPA, MCM complex, PreRC; delay replication initiation at origins
Additional RPA activity - Ub
- In addition to ATRIP and ATR, RPA at ssDNA recruits Rad6-Rad18 for mono-ubiquitination of PCNA
- Replicative pols have reduced affinity for Ub PCNA, dissociate
- TLS polsdo have affinity -> recruited to fork, resume DNA synthesis
- Error prone but allows progression
Sensor for DSBs, activation of ATM
- MRN (Has Mre11, Rad50, Nbs1)
- Interacts w/ DNA at break, may hold broken ends together
- MRN recruits ATM (normally inactive dimer), which auto-phosphorylates and activates
Activated ATM, phosphorylation of H2AX
- Phosphorylates targets to modulate multiple aspects of DNA metabolism/cell cycle control (~700 targets)
- Activated ATM targets H2AX histone (present in 10-15% of nucleosomes), phosphorylates
- pH2AX phosph. MDC1
-> recruits additional MRN, ATM
-> signal amplification - Key effectors of activated complex: CHK2, p53, DSB repair pathway proteins (BRCA1, CtlP, 53BP1)
Pathways for DSB repair and when they are applied
- Non-homologous end joining (simply re-joins ends, predominant in non-dividing cells; G1)
- Homologous recombination (uses hom. DNA as template, primarily used in late S phase/G2 when sister chromatid still available for copying)
KU protein structure and activity
- KU70, KU80 heterodimer
- Very abundant, binds strongly to ends of DSBs
- Vert.s: Recruits DNA-PKCS
- This recruits proteins to join broken ends (by NHEJ)
- Leads to some loss of information at DSB site
NHEJ pathways
- Nuclease digestion removing a few nts, then ligation
-> frameshift - Resection to expose ssDNA, alignment at region of microhomology, trimming of overhangs and ligation
-> Sequence loss either way; mutagenic
Conservation in DNA damage repair
- Many eukaryotic DNA damage response proteins are conserved, despite different naming
- The only exception is proteins which are involved w/ apoptosis, single single-celled organisms DO NOT participate
