Week 5 L3: RECOMBINATION mechanisms

Question 1

What are the steps in Robin hollidays model of recombination?

Answer 1

initial cleavage event 
strand invasion
Holliday Junction 
branch migration 
Resolution

Question 2

What is resolution?

Answer 2

nuclease specific for the junction and cleaves 2 of the starnds to go back to 2 duplexes. But there is red and green in the strands.
with a section of heteroduplex

Question 3

How can you get a double stand break (Initial break) ?

Answer 3

ionising radiation smashes its way through DNA

Question 4

How do you repair a double stand break?

Answer 4

recombination

Question 5

What enzyme is involved in exposing the 3’ end of double stranded DNA break?

Answer 5

Exonuclease

Question 6

What is the role of exonuclease?

Answer 6

Creates a ss 3’ gap

Question 7

What is a feature of the 3’ end?

Answer 7

invade the other chromosome stand (red stand)

Question 8

How does DNA replication (repair synthesis) work in recombination?

Answer 8

The heavy green stand forms a stand invasion D-loop with the heavy red stand
Red stand acts as a DNA template for the heavy green stand to grow.
The light green stand also uses the heavy red stand to repair
2 holiday junctions

Question 9

What is another way to generate a holliday junction?

Answer 9

chicken foot

Question 10

What are the 3 ways a holliday junction can be produced?

Answer 10

Recombination via stand invasion of the 3’end

DNA Repair double stand breaks

Replication fork reversion

All involve a recombination mechanism.

Question 11

What 2 steps in Holliday mechanism are needed to create a junction (1&2)?
Prejunction formation phase)

Answer 11

Creation of recombinogenic end (split in DNA)

Strand invasion

(prejunction formation phase)
–> junction

Question 12

What enzymes are involved in creating a recombinogenic end?

Answer 12

Nuclease

made of 3 proteins RecBCD

Question 13

What enzyme is involved in the stand invasion step?

Answer 13

protein RecA

junction

Question 14

What enzyme helps with directionality of branch migration?

Answer 14

RuvAB

Question 15

What enzyme helps with resolution of the strands ?

Answer 15

RuvC

Nuclease

Question 16

What activity does RecCBD have?

Answer 16

helicase and nuclease

Question 17

What are the functions of the individual proteins in Rec CBD?

Answer 17

RecB 3’ to 5’ helicase and nuclease
RecD 5’ to 3’ helicase
RecC recognises Chi
strand

All use ATP hydolysis to drive itself along the DNA, motor protein.

Question 18

How does RecB and Rec D differ in their degradation on DNA?

Answer 18

3’ - 5’ completely degrades

5’ - 3’ intermittently degrades.

Question 19

What type of proteins are the RecBCD?

Answer 19

motor proteins

Question 20

What does RecC do?

Answer 20

Scans for Chi seq

Question 21

What is the Chi seq?

Answer 21

crossover hotspot instigator sequence

Question 22

What happens when Chi is detected?

Answer 22

Changed confo of the RecBCD complex
stops degrading the 3’ stand and starts to degrade the 5’ stand more.
generates a 3’ end
can bind the next protein RecA

Question 23

What explains the polarity of the helicases?

Answer 23

The 5’ DNA starand goes into the RecD and the 3’ strand goes into the RecB

Question 24

What helps separate the 2 strands of DNA in RecB?

Answer 24

Pin structure so the helicase can pull these strands apart

Question 25

What are the 2 ways in which the 3' end on DNA can pass? | Alternative exits

Answer 25

path 1: avoids active site of the enzyme an dis not degraded | path 2: through active site of RecB and is degraded

Question 26

What are the domains of RecB?

Answer 26

Helicase domain which is bound to DNA and Nuclease domain and centre of the active centre

Question 27

What are the features of the active centre of RecB?

Answer 27

Metal ion bound to a number of acidic residues (ASp and Glu) | The 5'/3' end comes in from one side or the other

Question 28

What feature of the RecB helps cleave DNA?

Answer 28

Metal ion in the active centre

Question 29

What drives the enzyme along DNA duplex?

Answer 29

BIPOLAR BD helicase motors.

Question 30

What is the summary of the RecBCD mechanism?

Answer 30

3' end through active site, which excludes the 5' tail (not degraded so much) . Chi is encountered and the 3' end takes the other path and is no longer degraded and forms a ssDNA loop creating a recombinant end and 5' end degraded more fully AND can then bind Rec A

Question 31

Where does Chi bind in RecC?

Answer 31

Scanning site

Question 32

How does RecC prevent further digestion?

Answer 32

tightly binds 3'end and created a ssDNA loop

Question 33

What structure in RecB helps separate strands of DNA?

Answer 33

Pin