Eukaryotic DNA Replication and Genome Stability Flashcards
Structure
- Replication: what goes wrong and how to solve it
- Replication: proofreading polymerases
- RER
- MMR
- Fork stalling
- Fork breakage
- dNTP maintenance
What can go wrong during replication?
1) copying mistakes (from an undamaged template)
2) copying a damaged template
3) incorporating damaged nts
4) replication fork blockage
Solutions to combat replication error
1) high fidelity polymerases
2) proofreading polymerases (delta, epsilon)
3) translesion polymerases (bypass damage)
4) repair
5) post-replication repair (MMR, RER)
6) checkpoint mechanisms
7) apoptosis
DNA replication fork
- leading strand: Pol epsilon 5’ -> 3’
- lagging strand: Pol delta 3’ -> 5’ (Okazaki fragments)
- Pol alpha: primer
Okazaki fragment
- 200bp
- Eukarya
Pol alpha
- primase
- 10^3-10^4 fidelity
- low processivity
- PCNA-independent
Pol delta
- 3’ exonuclease
- 10^4-10^6 fidelity
- low processivity
- highly PCNA-dependent
Pol epsilon
- 3’ exonuclease
- 10^5-10^6 fidelity
- high processivity
- PCNA-dependent
fidelity estimates
- based on in vitro data
- in vivo accuracy may be >10^7
high selectivity for correct nt
polymerase pocket is tight
induced fit
- conformation change arises from catalytic addition of the correct nucleotide to the 3’OH
- if no conformational change, incorrect nucleotide leaves pocket
Potential misincorporation outcomes
1) dissociation (extrinsic proofreading exqnuclases remove the incorrect nt)
2) proofreading (nascent terminus flips to exo; incorrect nt removed)
3) extension (continued synthesis; mutation)
How do proofreading enzymes work?
flipping crosses the 35A distance to the exonuclease AS
Proofreading enzymes and cancer predisposition
- inherited mutations in Pol delta + epsilon exonuclease domains
- somatic tumours: Pol e exonuclease domain (10% in colon and endometrial cancers)
rNTP misincorporation
frequent, because they are 30-350x higher than dNTPs, and polymerases can be defective in their discrimination and proofreading
rNTP misincorporation rate
- alpha: 1/625
- delta: 1/5000
- epsilon: 1/1250
ramifications of ribonucleotide incorporation
- decreased stability
- fork stalling
Explain the decreased stability of ribonucleotide incorporation
- rna is less stable than dna
- spontaneous hydrolysis develops a “dirty” ss nick
- non-ligatable cyclic phosphate
explain the fork stalling consequence of ribonucleotide incorporation
- inefficient copying
- 60% bypass efficiency
- breakage
RER - the basics
- necessary (in mice)
- Aicaroi-Gautieres syndrome (partial defectivity in humans)
RER - the mechanism
1) Rnase H2 makes 5’ incision
2) Pol delta extension
3) Fen1 flap cleavage
4) nick ligation
MMR - the basics
- removes incorrectly inserted nucleotides
- improves replicative fidelity by ~100%
- case study: Lynch syndrome (increased risk of colon cancer)
- critical that parental strand is recognised as correct sequence (signal provided by PCNA/rNTPs/nicks)
MMR mechanism
1) Exo1 digests ssDNA around mismatch
2) Pol delta resynthesis
3) ligation
What happens when the replicative polymerases encounter a damaged leading template?
they stall!
- lagging template can still be reprimed by alpha
