Lecture 13 - Biochemistry of Homologous Recombination

Question 1

When does a cell carry out homologous recombination?

Answer 1

DNA double-strand breakds
DNA fragment
DNA single-strand gap

Question 2

Single strand gap repair - what is RecFOR?

Answer 2

RecA is loaded onto the strand by either recF, recO or recR

Question 3

How is a single strand gap repaired?

Answer 3

One strand has a gap, the other is full.

RecFOR loads RecA onto the gap which catalyses invasion by the broken chromosome into the non-broken one. This forms a Holliday junction which is cut by a third protein revC.

This leads to repair of full chromosome.

Question 4

What proteins cover Holliday junctions?

Answer 4

RevA and RevB

Question 5

What happens during double strand break?

Answer 5

The ends of the break are recognised by RecBCD which will load onto the DNA and form a single stranded part of DNA at a chi site which is then covered by recA.

RecA can then identify homologous regions for invasions.

Question 6

How many pathways of loading RecA?

Answer 6

Two - single strand break = loaded by RecFOR
double stranded break = loaded by RecBCD

Question 7

What does RecA do?

Answer 7

Invade full strands for DNA copying

Question 8

What is the action of RecBCD

Answer 8

RecBCD will recognise a double stranded break and use its translocase activity to open the DNA and translocate the DNA.

Question 9

RecBCD actions with Chi sites?

Answer 9

RecBCD will degrade both strands until they reach a chi site (TCC TGG TGG). When recBCD encounters this its activity changes by stopping degradation of the 3’ end but not the 5’ end. It will also load the recA protein on the single strand being formed.

Question 10

What does RecA form when it has been put on a single strand of DNA?

Answer 10

RecA protein filament

Question 11

What does the recA protien filament do?

Answer 11

looks for homolgous chromosomes to invade

Question 12

How many subunits is there and what does each do?

Answer 12

3
RecC = chi scanning site
Rec B = helicase and nuclease
Rec D = helicase

Question 13

Before chi site is the DNA closed or open?

Answer 13

Open

Question 14

What happens to the RecBCD subunits once its recognised chi?

Answer 14

RecC still recognises Chi
Rec B = opens DNA
Rec D = no longer works

Question 15

does RecA coat single stranded DNA?

Answer 15

Yes

Question 16

How does RecA form a nucleoprotein filament?

Answer 16

Single stranded DNA is covered by the protien SSB.
RecA is loaded on a few molecule which is called nucleation.

Question 17

Is nucleation slow?

Answer 17

Yes before picking up speed and extending throughout the molecule.

Question 18

What happens after RecA find homologous regions to invade after the recA protein filament coats the 2 DNA strands?

Answer 18

The single strand enters the double strand by invasion and make a D loop this triggers synthesis of destroyed DNA and recpature of the other end.

Question 19

How does RecA test to see if something is homologous?

Answer 19

RecA catalyses short contact between the ss and the other chromosomes in a random matter. They then undergo intersegmental sampling which allows it to test 2 peices of DNA and the same time and can also slide along the DNA.

Question 20

What happens when RecA finds a homologous region?

Answer 20

All contacts are short lives unless it is homologous and then a base pairing occurs between SS and one strands of the chromosome = this is the D loop and stabilises the pairing.

Question 21

After stabilising recA and the DNA do a DNA strand exchange, what is this?

Answer 21

Originally the RecA forms a three stranded intermediate as it will sit between the 2 ds strands. A duplex is formed here with one strand from the original chromosome and one strand from the other. This free’s up one strand to create a branch.

Question 22

How does the branch move and what does this do?

Answer 22

This branch moves via branch migration extending the exhange of strand from the intial double strand to a mixed strand.

Question 23

What happens after the invasion has taken place and strand invasion?

Answer 23

RecA needs to disocciate and the synthesis of the missing nucleotides must happen. Meanwhile the Dloop grows until it reaches the other strand = this can then be fixed. This makes Holliday junctions.

Question 24

What recognises Holliday junctions?

Answer 24

Ruvabc

Question 25

What does RuvABC do to Holliday junctions?

Answer 25

Moves and cleaves them leading to 2 repaired chromosomes

Question 26

How do Holliday junctions move (branch migration)?

Answer 26

They can go left or right

Question 27

Where can Holliday junctions be cut?

Answer 27

Either left to right or up and down (redraw if you are cutting up and down cause its more confusing)

Question 28

How would you redraw the molecule to make it easier to understand?

Answer 28

Instead of - make it -
x ]
x [
- -
Then push the junctions to make a big cross

Question 29

What proteins are important in stabilising Holliday junctions?

Answer 29

RuvA and RuvB

Question 30

What does Ruv A do?

Answer 30

Holds the junction in a square planar cinfiguraton e.g. stop it bending

Question 31

What does RuvB do?

Answer 31

Directs branch migration

Question 32

What happens to RuvB once branch migration has happened?

Answer 32

it will be able to cut in the A+B plane or the A’ + B’ plane