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L3 Gene Regulation > Week 5 L3: RECOMBINATION mechanisms > Flashcards

Week 5 L3: RECOMBINATION mechanisms Flashcards

1
Q

What are the steps in Robin hollidays model of recombination?

A 
initial cleavage event 
strand invasion
Holliday Junction 
branch migration 
Resolution
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2
Q

What is resolution?

A

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

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3
Q

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

A

ionising radiation smashes its way through DNA

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4
Q

How do you repair a double stand break?

A

recombination

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5
Q

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

A

Exonuclease

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6
Q

What is the role of exonuclease?

A

Creates a ss 3’ gap

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7
Q

What is a feature of the 3’ end?

A

invade the other chromosome stand (red stand)

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8
Q

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

A

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

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9
Q

What is another way to generate a holliday junction?

A

chicken foot

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10
Q

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

A

Recombination via stand invasion of the 3’end

DNA Repair double stand breaks

Replication fork reversion

All involve a recombination mechanism.

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11
Q

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

A

Creation of recombinogenic end (split in DNA)

Strand invasion

(prejunction formation phase)
–> junction

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12
Q

What enzymes are involved in creating a recombinogenic end?

A

Nuclease

made of 3 proteins RecBCD

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13
Q

What enzyme is involved in the stand invasion step?

A

protein RecA

junction

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14
Q

What enzyme helps with directionality of branch migration?

A

RuvAB

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15
Q

What enzyme helps with resolution of the strands ?

A

RuvC

Nuclease

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16
Q

What activity does RecCBD have?

A

helicase and nuclease

17
Q

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

A

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.

18
Q

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

A

3’ - 5’ completely degrades

5’ - 3’ intermittently degrades.

19
Q

What type of proteins are the RecBCD?

A

motor proteins

20
Q

What does RecC do?

A

Scans for Chi seq

21
Q

What is the Chi seq?

A

crossover hotspot instigator sequence

22
Q

What happens when Chi is detected?

A

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

23
Q

What explains the polarity of the helicases?

A

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

24
Q

What helps separate the 2 strands of DNA in RecB?

A

Pin structure so the helicase can pull these strands apart

25
Q

What are the 2 ways in which the 3’ end on DNA can pass?

Alternative exits

A

path 1: avoids active site of the enzyme an dis not degraded

path 2: through active site of RecB and is degraded

26
Q

What are the domains of RecB?

A

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

27
Q

What are the features of the active centre of RecB?

A

Metal ion bound to a number of acidic residues (ASp and Glu)

The 5’/3’ end comes in from one side or the other

28
Q

What feature of the RecB helps cleave DNA?

A

Metal ion in the active centre

29
Q

What drives the enzyme along DNA duplex?

A

BIPOLAR BD helicase motors.

30
Q

What is the summary of the RecBCD mechanism?

A

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

31
Q

Where does Chi bind in RecC?

A

Scanning site

32
Q

How does RecC prevent further digestion?

A

tightly binds 3’end and created a ssDNA loop

33
Q

What structure in RecB helps separate strands of DNA?

A

Pin

L3 Gene Regulation (14 decks)

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