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BIOL253 > L8: DNA damage tolerance and response > Flashcards

L8: DNA damage tolerance and response Flashcards

1
Q

How would they be repaired?…
- Mismatched base pairs
- Altered/absent bases

A
  • Replicated by usual replication machinery, resulting in mutation
  • Cannot be replicated normally; specific DNA pols can replicate, tolerating damage
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2
Q

What happens when normal replicative polymerases (III, delta, epsilon) try to replicate damaged bases (give examples of base damage)

A
  • 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
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3
Q

TLS DNA pols in E.coli and in humans (categorised by family)

A

E.coli
- Y-family: DNA pol IV, DNA pol V
Human
- Y-family: Pol eta, Pol iota, Pol kappa
- B-family: Pol zeta

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4
Q

How do TLS pols differ from normal DNA pols? (and what is the consequence?)

A
  • 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)
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5
Q

Interaction between TLS pols and sliding clamp

A
  • Y-family ‘little finger’ domain interacts with beta clamp or PCNA (depending on organism).
  • Domain contacts DNA close to lesion site
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6
Q

Levels of TLS pols

A
  • 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
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7
Q

What two scenarios lead to induction of DNA damage response?

A
  • 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
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8
Q

Inducing DNA damage response (first step + consequences)

A
  1. ssDNA and DNA DSBs are recognised by damage sensor proteins
  • Increased DNA repair proteins
  • Delayed cell cycle
  • Programmed cell death (only in multicellular organisms)
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9
Q

Key DNA damage response proteins (when inactive - bacteria)

A

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)

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10
Q

Key DNA damage response proteins (when active - bacteria)

A

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

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11
Q

Examples of genes under SOS regulation by LexA - bacteria

A
  • DinI
  • (NER) UvrA, B, D
  • (Recomb. repair) RecA, RuvA, RuvB
  • (TLS pols) pol IV, polV
  • SulA (inhibits cell division -> greater time window for repair)
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12
Q

Re-establishing repression of SOS genes after repair (3 drivers) - bacteria

A
  • 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
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13
Q

DNA damage response overview -Eukaryotes

A
  • 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)
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14
Q

RPA - Eukaroytes

A
  • 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
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15
Q

ATR activity (key processes w/ examples of targets)

A
  1. Cell cycle control
    e.g. CHK1, arrests cell cycle -> time window
  2. Replication fork stabilisation
    e.g. RPA, Pols, Rad17-Rfc, 9-1-1; slow replication fork progress
  3. Replication origin control
    e.g. RPA, MCM complex, PreRC; delay replication initiation at origins
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16
Q

Additional RPA activity - Ub

A
  • 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
17
Q

Sensor for DSBs, activation of ATM

A
  • 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
18
Q

Activated ATM, phosphorylation of H2AX

A
  • 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)
19
Q

Pathways for DSB repair and when they are applied

A
  1. Non-homologous end joining (simply re-joins ends, predominant in non-dividing cells; G1)
  2. Homologous recombination (uses hom. DNA as template, primarily used in late S phase/G2 when sister chromatid still available for copying)
20
Q

KU protein structure and activity

A
  • 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
21
Q

NHEJ pathways

A
  1. Nuclease digestion removing a few nts, then ligation
    -> frameshift
  2. Resection to expose ssDNA, alignment at region of microhomology, trimming of overhangs and ligation

-> Sequence loss either way; mutagenic

22
Q

Conservation in DNA damage repair

A
  • 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

BIOL253 (19 decks)

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