14. DNA replication and damage Flashcards
What are the 3 steps of DNA replication
DNA replication - duplication of genomic DNA:
1) Initiation
2) Elongation
3) Termination
Why is DNA replication said to be semi-conservative?
DNA replication semi-conservative because in the new ds DNA one strand is from previous ds DNA molecule (1 old strand + 1 new strand) - old strand acts as a template
What is the structure of an E. coli chromsome during replication?
E. coli chromsome - circular - replication start at OriC - bidirectional replication from OriC - replication termination across from OriC
What enzyme performs elongation in DNA replication?
Elongation in DNA replication - DNA pol III at replication fork - catalyzes new strand synthesis
What are the proteins + subunits of DNA pol III at replication fork?
DNA polymerase III:
- core polymerase
- beta-clamp
- clamp loader
Other enzymes:
- DnaB helicase
- DnaG primase
- DNA ligase
- DNA topoisomerase
- DNA pol I (Okazaki fragments)
- SSB proteins
What are the subunits fo DNA pol III and what are their functions?
What is required for DNA pol III to start elongation?
A primer at replication start site - short RNA primer - added by DNA primase => DNA pol III synthesises only 5’->3’
What is the function of the sliding clamp?
Sliding clamp behind DNA pol III - ensures 5’->3’ and stays in DNA strand
Explain in detail elongation process
Elongation in replication:
1) DNA primase adds RNA primer
2) DNA pol III binds - sliding clamp keeps behind DNA pol III
3) Leading strand elongation continuous, lagging strand elongation non-continuous in Okazaki fragments - lagging strand forms a loop because DNA pol III both form a dimer - move together
4) DNA pol I removes RNA primers + synthesises gaps
5) DNA ligase seals the gaps left
Why does the lagging strand form a loop during elongation?
Because DNA pol III on leading and lagging strand form a dimer - move physically together in the same direction - loop formed so can move into the same direction
What enzymes removes RNA primers?
DNA pol I removes RNA primers from lagging strand by exonuclease action+ synthesises DNA into gaps
What are the DNA polymerases involved in E. coli replication? What are their functions?
DNA pol III
DNA pol I
DNA primase - RNA polymerase
How is replication initiated in E. coli?
Initiation at origin or replication - OriC
1) OriC 2 components: 13-mer repeat + 9-mer repeat, addition of DnaA-ATP initiator proteins
2) Folding
3) Opening of OriC at 13-mer repeats by helicase DnaB
4) When ds separated - SBS proteins bind to prevent re-annealing
5) DNA primase binds to bind an RNA primer
6) DNA pol III is recruited - starts synthesis from primers
7) DNA topoisomerase (gyrase) keeps unwinding DNA supercoil
How is replication terminated in E. coli?
Replication termination:
1) Elongation happens bidirectionally on a circular chromosome - two DNA pol III meet - two replication forks meet across from OriC
2) Replication stopped at specific sites - ex left strand cannot go through terA - only right goes - meets
Why are there two replication forks on a single E. coli chromosome?
Replication in E. coli is bidirectional - two replication forks move in opposite directions from OriC - each replication fork replicates half of chromosome - meet opposite of OriC
What are the causes of replication stop?
Replications stops if:
1) Replication fork collapse
2) Replication fork reversal
-> needs to be re-started
What happens when a replication fork encounters a nick/gap in DNA?
Replication fork encounters nicks/gaps in DNA (ssDNA gap)
1) Detection of ssDNA gap
2) Induced dsDNA break
3) Replisome disassembled
=> pontential loss of genetic material
How is replication-dependent ds break (DSB) repaired?
dsDNA break induced by replication fork collapse is repaired:
1) RecBCD recruited to dsDNA break site
2) RecA loading
3) HR using homologs as template for synthesis of the break
4) HJ formation
5) HJ cleavage by RuvABC
6) PriA restarts replication at the site - away from OriC
How is replication re-started after a dsDNA break is repaired that caused replication fork collapse?
Replication re-started:
1) PriA recognises 3-way junctions
2) PriA binds - PriB + DnaT bind
3) DnaC (aids DnaB), DnaB (helicase) recruited
4) Replication away from OriC re-started
Does PriA only target replication structures?
No, PriA also targets recombinations structures:
1) Recognizes D-loop
2) PriA binds
3) PriB + DnaT recruited - bind
4) DnaC (aids DnaB), DnaB (helicase) recruited
5) Replication re-started away from OriC
What does PriA target?
PriA targets:
1) 3-way junctions - replications structure
2) D-loops - recombination structure
When does replication fork collapse happen?
Replication fork collapses when it runs into nick/gap in dsDNA
What enzymes are required to create D-loop?
DNA repair enzymes are required to create a D-loop (D-loop created when RecA coated strand invades homologous strand) - necessary for re-starting replication
Can E. coli replication be initiated not at OriC?
Yes - if replication fork collapses or reverses - PriA - initiator of replication away from OriC
What causes replication fork reversal?
Replication fork is reversed because of:
1) collision with transcription enzymes
2) replication defects
3) strong protein binding to DNA (ex SBS in the way)
-> replication fork stalled - downstream replication re-start
Why does replication fork reversal lead to DSB?
Replication fork reversal leads to ds breaks (DSB) because of RuvABC:
1) RuvAB recognises 3-way junction
2) RuvC cleaves - ds break created
How can replication fork be reversed by RecA?
Replication fork reversed:
1) RecA binds -> reverses replication
2) RuvAB recruited to 3-way junction - RuvAB binds
3) ds break created by RuvC
How can repair protein RecA create DNA breaks?
RecA can create DNA breaks when:
1) RecA binds - replication fork reversed
2) RuvAB recruited to 3-way junction - bind
3) RuvC recruited to RuvAB - RuvC cuts ds -> ds break created because RecA bound, cut by RuvC
What are the possible outcomes of replication fork reversal?
When replication fork reversed - RuvAB at 3-way junction (HJ):
1) RuvC cuts -> DSB -> RecBCD recognises -> RecA => repair by HR
2) RecBCD binds - exonuclease activity - degrades:
- large piece of DNA -> promotes recombination -> PriA => replication re-started
- small piece of DNA -> PriA => replication re-started
What are Ter sites?
Ter site = terminus binding site - protein binds to replication terminus on DNA
How can linear DNA be formed at Ter sites?
Tus-Ter complex formed:
1) Tus proteins bind at Ter sites - replication fork blocked - collision
2) Replication within the restrained region -> linear product
3) Second replication within restrained region -> linear product
=> Over replication - can be corrected by RecBCD
Summary of causes of replication arrest, their recombination proteins, origin of linear DNA and model
Which process, replication or recombination, creates problems and which repairs them?
Both replication adn recombination processes create problems but also repair them
Lecture summary
