Week 5 L4: RecA & RuvAB

Question 1

What is the function of RecA?

Answer 1

Create an end which can invade the opposing duplex.

strand invasion

Question 2

Does RecA polymerise on ss or ds DNA?

Answer 2

ss

Question 3

What does RecA produce?

Answer 3

A filament, the active species in strand exchange

Question 4

How much RecA does the filament contain?

Answer 4

1 monomer every 4-9 nt DNA in a stable complex

Question 5

Is ATP required to form filament?

Answer 5

NO but ATP increases dissociation

Question 6

Is the RecA filament polar?

Answer 6

YES bind in 5’ to 3’ direction, migrates to 3’ end of molecule

Question 7

What is the equivalent of RecA in eukaryotes?

Answer 7

RAD51 in yeast

Question 8

How is the RecA filament formed?

Answer 8

Many RecA binding to ssDNA

= RecA nucleofilament

Question 9

What does RecA promotoe?

Answer 9

Homologous pairing

Question 10

What then binds when the RecA nucleofilament is formed?

Answer 10

homologous DNA duplex

homologous alignment

Question 11

What happens after Homologous alignment occurs?

Answer 11

strand exchange occurs, mediated by the RecA protein

Question 12

What occurs when strand exchange occurs?

Answer 12

a D loop
hybridisation between the green invading strands and in one of the strands of the duplex with displacement of the other strand.

Question 13

What enzyme is involved in branch migration on a junction?

Answer 13

2 proteins, RuvA and RuvB

Question 14

Why are enzymes needed in branch migration?

Answer 14

spontaneous branch migration is slow, must be catalysed.

accelerated by p

Question 15

What are the features of RuvA?

Answer 15

binds selectively to DNA junctions as a tertramer

Question 16

What are the features of RuvB?

Answer 16

Binds DNA as hexamer
Exhibits DNA helicase activity.
ATP dependent helicase

Question 17

What is RuvAB encoded on?

Answer 17

Ruv operon of E.coli

Question 18

Does the RuvA have an electropositive and electronegative side?

Answer 18

YES

Question 19

What is the electropositive side of RuvA abundant in?

Answer 19

• NH2 terminus

- abundant in arg and lys

Question 20

Is the electropositive RuvA good at binding DNA?

Answer 20

YES

Question 21

What conformation does DNA have when binding to RuvA?

Answer 21

DNA open junction
flat form
open square conformation

Question 22

When does the flat form of DNA occur?

Answer 22

when no metal ion is present.

Question 23

What does RuvA do to the structure of the holliday junction?

Answer 23

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

Question 24

How do you increase branch migration?

Answer 24

lower the ionic strength.

easier to break base pairs and rehybridize them at the centre, of the centre is open.

Question 25

What is the structure of RuvB?

Answer 25

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.

Question 26

Where does dna pass through in the RuvB?

Answer 26

The Dna sized hole in the centre of the ring

Question 27

How does RuAB complex work on a junction?

Answer 27

• 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.

Question 28

What is the main way RuvA facilitates Branch migration?

Answer 28

Changes the strcuture of DNA

Question 29

What is the main way RuvB facilitates Branch migration?

Answer 29

accelerating the process and conferring directionality by pulling on the DNA. using its energy from Atp HYDROLYSIS.

Question 30

What is the direction of DNA through the RuvAB complex?

Answer 30

Comes in through the top (RuvA) and pulled out at the sides (RuvB) through its pumping-like action