Lecture 8 - replication forks and BIR

Question 1

What can occur when replication forks get stuck?

Answer 1

• resume
• fall apart
• start again from beggining

Question 2

In which direction does extension and synthesis occur on DNA strand?

Answer 2

5’ to 3’

3’ end extended

Question 3

What is formed when RF hits a nick?

Answer 3

Single-end DSB

Question 4

What are single end DSBs bad?

Answer 4

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

Question 5

What happens when a second RF arrives at the other side of a site where a previous RF has collapsed?

Answer 5

DSB is generated and leading to normal mechanisms of repair

-ends can invade intact chromatids and be repaired by normal DSB repair

Question 6

What are the five causes of blocks a RF may face?

Answer 6

• 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)

Question 7

What are the four ways RNA polymerase can block RF?

Answer 7

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

Question 8

What happens when there is DNA damage on the lagging strand in front of RF?

Answer 8

primer can be laid down at site of damage but not extended, nick is avoided and can be fixed when RF moves away

Question 9

What happens when there is DNA damage on leading strand in front of the replication fork?

Answer 9

Either replication continues by repriming of the leading strand (rare) or gap formed in the leading strand

Question 10

What are the 4 ways replication forks can be restored?

Answer 10

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)

Question 11

What is the mechanism of 1-Fork regression, template switch, reversal, laid over?

Answer 11

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

Question 12

What is the mechanism of 2-Fork regression, removal of block, degredation of extruded dsDNA, second attempt?

Answer 12

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

Question 13

Why is the second attempt mechanism problematic?

Answer 13

need different sorts of nucleases to degrade 5’ to 3’ and 3’ to 5’

Question 14

What is break induced replication?

Answer 14

A mechanism of homologus recombination

When fork regression results in single-ended DSBs and is followed by strand invasion and continuation of replication

Question 15

What is the mechanism of 3-Fork regression HJ cleavage, BIR?

Answer 15

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

Question 16

How is BIR method of replication restart different?

Answer 16

leads to establishment of new RF

Question 17

What is the mechanism of 4-fork regression, 3’ends of extruded, invasion, resolution of dHJ?

Answer 17

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

Question 18

What happens when cells are exposed to a lot of UV radiation?

Answer 18

lots of dsbreaks

Question 19

What is Deinococcus radiodurans resistant to?

Answer 19

Ionising radiation, gamma rays
UV light
Cross linking reagents
Desication

Question 20

How does mitomycin C act?

Answer 20

-crosslink guanosine residues by binding to guanosines on opposide or adjacent strands
leads to
-interstrand crosslinking
-intrastrand crosslinking

Question 21

How is Deinococcus radiodurans reassembled?

Answer 21

using SDSA on long stretches of DNA - ESDSA

Question 22

How was the kinetics of DSB repair shown in D.radiodurans?

Answer 22

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

Question 23

In D.radiodurans what role does RecA have?

Answer 23

plays important role in efficient genome reassembley

Question 24

How does the repair process in D radiodurans occur?

Answer 24

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

Question 25

How was it shown that D radiodurans uses ESDSA not SSA?

Answer 25

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

Question 26

Why has D radiodurans evolved to be radiation tolerant?

Answer 26

-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

Question 27

Why is D radiodurans better then e.coli at scavenging ROS?

Answer 27

better at producing enzymes to remove highly reactive oxygen molecules to protect proteins

Question 28

Why is a high Mn/Fe ratio correlated with high radioation resistance and low protein oxidation?

Answer 28

Mn acts as an oxygen scavger

Question 29

What happens when E’coli is supplemented with small molecules found in Dradiodurans?

Answer 29

E.coli resist ionising radiation with addition of

• Uridine
• Manganese
• Phosphate
• > form a complex to protect against ionising radiation

Question 30

What does Deinococcus radiodurans have to protect it?

Answer 30

• 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