Week 5 L4: RecA & RuvAB Flashcards
What is the function of RecA?
Create an end which can invade the opposing duplex.
strand invasion
Does RecA polymerise on ss or ds DNA?
ss
What does RecA produce?
A filament, the active species in strand exchange
How much RecA does the filament contain?
1 monomer every 4-9 nt DNA in a stable complex
Is ATP required to form filament?
NO but ATP increases dissociation
Is the RecA filament polar?
YES bind in 5’ to 3’ direction, migrates to 3’ end of molecule
What is the equivalent of RecA in eukaryotes?
RAD51 in yeast
How is the RecA filament formed?
Many RecA binding to ssDNA
= RecA nucleofilament
What does RecA promotoe?
Homologous pairing
What then binds when the RecA nucleofilament is formed?
homologous DNA duplex
homologous alignment
What happens after Homologous alignment occurs?
strand exchange occurs, mediated by the RecA protein
What occurs when strand exchange occurs?
a D loop
hybridisation between the green invading strands and in one of the strands of the duplex with displacement of the other strand.
What enzyme is involved in branch migration on a junction?
2 proteins, RuvA and RuvB
Why are enzymes needed in branch migration?
spontaneous branch migration is slow, must be catalysed.
accelerated by p
What are the features of RuvA?
binds selectively to DNA junctions as a tertramer
What are the features of RuvB?
Binds DNA as hexamer
Exhibits DNA helicase activity.
ATP dependent helicase
What is RuvAB encoded on?
Ruv operon of E.coli
Does the RuvA have an electropositive and electronegative side?
YES
What is the electropositive side of RuvA abundant in?
- NH2 terminus
- abundant in arg and lys
Is the electropositive RuvA good at binding DNA?
YES
What conformation does DNA have when binding to RuvA?
DNA open junction
flat form
open square conformation
When does the flat form of DNA occur?
when no metal ion is present.
What does RuvA do to the structure of the holliday junction?
opens the structure to the plus looking one
Stacked X form to unfolded confo (as if the metal ion had not folded the structure)
Extended open form
How do you increase branch migration?
lower the ionic strength.
easier to break base pairs and rehybridize them at the centre, of the centre is open.
What is the structure of RuvB?
2 rings f proetin
each ring has 6 molecules of RuvB
Hexamer of RuvB forming a ring adjacent to another hexameeic ring.
Open centre where DNA can pass through this.
Where does dna pass through in the RuvB?
The Dna sized hole in the centre of the ring
How does RuAB complex work on a junction?
- RuvA tetramer targets the junction and forces the open square form.
- On either side we have the RuvB hexamers which are ATP dependent helicases, acts as a pump. Pulls the DNA through the hexameric ring.
What is the main way RuvA facilitates Branch migration?
Changes the strcuture of DNA
What is the main way RuvB facilitates Branch migration?
accelerating the process and conferring directionality by pulling on the DNA. using its energy from Atp HYDROLYSIS.
What is the direction of DNA through the RuvAB complex?
Comes in through the top (RuvA) and pulled out at the sides (RuvB) through its pumping-like action
