Lecture 8 - replication forks and BIR Flashcards
What can occur when replication forks get stuck?
- resume
- fall apart
- start again from beggining
In which direction does extension and synthesis occur on DNA strand?
5’ to 3’
3’ end extended
What is formed when RF hits a nick?
Single-end DSB
What are single end DSBs bad?
risk of ectopic recombination as can interact with outher places in the genome if there isn’t a homolgous region nearby - leading to genome rearrangements
What happens when a second RF arrives at the other side of a site where a previous RF has collapsed?
DSB is generated and leading to normal mechanisms of repair
-ends can invade intact chromatids and be repaired by normal DSB repair
What are the five causes of blocks a RF may face?
- nicks (ssbreaks)
- pyrimidine dimers produced by UV (crosslinking between 2 adjacent pyrimadine dimers, T and C)
- bulky base modifications
- interstrand crosslinks (e.g. mytocin C)
- transcription complexes (RNA polymerase)
What are the four ways RNA polymerase can block RF?
1) RNA polymerase already on DNA but is slower, unavoidable
2) RNApolymerase may be working in the opposite direction to RF, stops because too many supercoils in intervening DNA
3) RNA polymerase hit DNA damage ahead of it, sticking it in place
4) many polymerases may have been acting in sequence leading to a roadblock at damaged DNA, RF collapses and removed
What happens when there is DNA damage on the lagging strand in front of RF?
primer can be laid down at site of damage but not extended, nick is avoided and can be fixed when RF moves away
What happens when there is DNA damage on leading strand in front of the replication fork?
Either replication continues by repriming of the leading strand (rare) or gap formed in the leading strand
What are the 4 ways replication forks can be restored?
1- Fork regression, template switch, reversal, new leading strand laid over site of damage
2 - Fork regression, removal of block, degredation of extruded dsDNA, RF restored second attempt to replicate
3 - Fork regression, HJ cleavage, BIR (single ended dsbreak)
4 - Fork regression, produced 3’ ends of extruded DNA, invasion DNA on the other side, resolution of dHJ (no dsbreaks)
What is the mechanism of 1-Fork regression, template switch, reversal, laid over?
1) Lesion on the leading strand, lagging strand temporarily continues until cannot move
2) regression of the replication fork with helicases, reforms pairs of the template strand
3) synthesised lagging strand is separated and available, if regression fork goes back enough leading strand can complementary bind to the newly syntheised lagging strand
4) leading strand uses lagging strand as template to extend 3’ end ‘template switch’
5) newly synthesised leading strand from lagging strand template laid over damage, backwards movement reversed and continuation of the RF
- whether or not has been repaired
What is the mechanism of 2-Fork regression, removal of block, degredation of extruded dsDNA, second attempt?
1) block on both strands leads to formation of the regression fork
2) allows repair enzymes to repair the damage
3) exonucleases degrade extruded short dsDNA
4) regression fork reversed and can continue as replication fork
Why is the second attempt mechanism problematic?
need different sorts of nucleases to degrade 5’ to 3’ and 3’ to 5’
What is break induced replication?
A mechanism of homologus recombination
When fork regression results in single-ended DSBs and is followed by strand invasion and continuation of replication
What is the mechanism of 3-Fork regression HJ cleavage, BIR?
1) Replication fork regression produces a Holliday junction
2) cleavage of HJ in symmetric cuts produces an intact template and a broken ended molecule
3)5’ to 3’ econuclease resection leads to 3’ overhang
4) RecA recombinase binds to ssDNA 3’overhang and begins a homology search
5) results in strand invasion and creation of D loop
6)results in reestablishment of RF allowing lagging and leading strand synthesis
HOWEVER
7)other side of the D loop is crossed
8) branch migration allows gap to be filled in
9) holliday junction is formed by ligation of strands
10) HJ resolved by resolvases and sister chromatids separated
How is BIR method of replication restart different?
leads to establishment of new RF
What is the mechanism of 4-fork regression, 3’ends of extruded, invasion, resolution of dHJ?
1) RF arrives and arrests at the block
2) RF regression with a ds extruded, block repaired repair enzymes
3) 5’ to 3’ exonuclease exposes 3’ tail
4) 3’ tail joins to complementary sequences nearby
5) forms a D loop and dHJ
6) cleavage of dHJ and RF restart
What happens when cells are exposed to a lot of UV radiation?
lots of dsbreaks
What is Deinococcus radiodurans resistant to?
Ionising radiation, gamma rays
UV light
Cross linking reagents
Desication
How does mitomycin C act?
-crosslink guanosine residues by binding to guanosines on opposide or adjacent strands
leads to
-interstrand crosslinking
-intrastrand crosslinking
How is Deinococcus radiodurans reassembled?
using SDSA on long stretches of DNA - ESDSA
How was the kinetics of DSB repair shown in D.radiodurans?
1) cells were exposed to gamma irradiation
2) left to recover for different times
3) DNA isolated and cut with restriction endonuclease NotI (easier to get fragments on gel)
4) analysis on agarose gel
5) showed that if left long enough, pattern from not gamma radiated cells restored
conclusion: D.radiodurans is able to repair genome after severe fragmentation
In D.radiodurans what role does RecA have?
plays important role in efficient genome reassembley
How does the repair process in D radiodurans occur?
1) genome cut and 3’ overhangs liberated
2) repair process starts by recombinase searching for complementary pairing of 3’ ends
3) long sections of DNA synthesised meaning ends can anneal and overhangs removed
4) chromosomes reestablished by HR and HJ formation
How was it shown that D radiodurans uses ESDSA not SSA?
using heavy and light DNA strands
For SSA predicted that semiconservative repair
For ESDSA predicted to have conservative
Conservative shown, with regions of H/H and L/G
Why has D radiodurans evolved to be radiation tolerant?
-resistant to 4000 Gy, naturally on earth no more than 0.2 Gy
-radiation tolerant bacteria often found in desert soils -> indicates that radiation tolerence is linked to dessecation tolerance
how? dehydration linked to oxidative damage of proteins
Why is D radiodurans better then e.coli at scavenging ROS?
better at producing enzymes to remove highly reactive oxygen molecules to protect proteins
Why is a high Mn/Fe ratio correlated with high radioation resistance and low protein oxidation?
Mn acts as an oxygen scavger
What happens when E’coli is supplemented with small molecules found in Dradiodurans?
E.coli resist ionising radiation with addition of
- Uridine
- Manganese
- Phosphate
- > form a complex to protect against ionising radiation
What does Deinococcus radiodurans have to protect it?
- 4 to 10 copies of genome per cell
- efficient DNA repair system
- system to eliminate ROS -> protecting proteins
- system to destroy damaged nucleotides
- system to export short DNA fragments, degrade them outside cell and recycle undamaged nucleotides
