Week 5 L3: RECOMBINATION mechanisms Flashcards
What are the steps in Robin hollidays model of recombination?
initial cleavage event strand invasion Holliday Junction branch migration Resolution
What is resolution?
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
How can you get a double stand break (Initial break) ?
ionising radiation smashes its way through DNA
How do you repair a double stand break?
recombination
What enzyme is involved in exposing the 3’ end of double stranded DNA break?
Exonuclease
What is the role of exonuclease?
Creates a ss 3’ gap
What is a feature of the 3’ end?
invade the other chromosome stand (red stand)
How does DNA replication (repair synthesis) work in recombination?
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
What is another way to generate a holliday junction?
chicken foot
What are the 3 ways a holliday junction can be produced?
Recombination via stand invasion of the 3’end
DNA Repair double stand breaks
Replication fork reversion
All involve a recombination mechanism.
What 2 steps in Holliday mechanism are needed to create a junction (1&2)?
Prejunction formation phase)
Creation of recombinogenic end (split in DNA)
Strand invasion
(prejunction formation phase)
–> junction
What enzymes are involved in creating a recombinogenic end?
Nuclease
made of 3 proteins RecBCD
What enzyme is involved in the stand invasion step?
protein RecA
junction
What enzyme helps with directionality of branch migration?
RuvAB
What enzyme helps with resolution of the strands ?
RuvC
Nuclease
What activity does RecCBD have?
helicase and nuclease
What are the functions of the individual proteins in Rec CBD?
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.
How does RecB and Rec D differ in their degradation on DNA?
3’ - 5’ completely degrades
5’ - 3’ intermittently degrades.
What type of proteins are the RecBCD?
motor proteins
What does RecC do?
Scans for Chi seq
What is the Chi seq?
crossover hotspot instigator sequence
What happens when Chi is detected?
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
What explains the polarity of the helicases?
The 5’ DNA starand goes into the RecD and the 3’ strand goes into the RecB
What helps separate the 2 strands of DNA in RecB?
Pin structure so the helicase can pull these strands apart
What are the 2 ways in which the 3’ end on DNA can pass?
Alternative exits
path 1: avoids active site of the enzyme an dis not degraded
path 2: through active site of RecB and is degraded
What are the domains of RecB?
Helicase domain which is bound to DNA and Nuclease domain and centre of the active centre
What are the features of the active centre of RecB?
Metal ion bound to a number of acidic residues (ASp and Glu)
The 5’/3’ end comes in from one side or the other
What feature of the RecB helps cleave DNA?
Metal ion in the active centre
What drives the enzyme along DNA duplex?
BIPOLAR BD helicase motors.
What is the summary of the RecBCD mechanism?
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
Where does Chi bind in RecC?
Scanning site
How does RecC prevent further digestion?
tightly binds 3’end and created a ssDNA loop
What structure in RecB helps separate strands of DNA?
Pin
