Week 5 L5: RuvC

Question 1

What is the function of RuvC?

Answer 1

the junction gets resolved back into 2 duplexes. Required for all methods of recombination.

Question 2

What is the main player in the resolution of junction?

Answer 2

RuvC

dimeric nuclease, specific for the structure of the 4-way junction.

Question 3

How many cleavage events occur in resolution?

Answer 3

2, in orderto separate 2 duplexes from the junction

Question 4

What enzyme cleaves the four-way junction in bacteriophage infected bacteria?

Answer 4

T4 endonuclease VII

Question 5

Where does the T4 endonuclease VII cleave?

Answer 5

2 cleavages symmetrically disposed about the junction. 2 nucleotides to the point of strand exchange.

Question 6

What is the human version of RuvC?

Answer 6

GEN1

Question 7

Are these enzymes basic or acidic?

Answer 7

BASIC

lots or Arg and Lys residues so it can bind to DNA covered in phosphate groups

Question 8

Is RuvC dimeric or trimeric?

Answer 8

Dimeric

binds as a dimer (GEN1 too)

Question 9

How many junction resolving activities are there in humans?

Answer 9

multiple

As this is a really important process and cant rely on just 1 activity

Question 10

How are these resolution activities separated in humans?

Answer 10

spatially and temporally (different parts of the cell cycle)

Question 11

What are the enzymes in G2/M phase in humans?

Answer 11

SLX1-SLX4-MUS81-EME1

Question 12

What enzyme is present in anaphase in humans?

Answer 12

GEN1

Question 13

Where is GEN1 present?

Answer 13

As it can only reach the nucleus when the nuclear membrane breaks down

Question 14

What enzyme is present in cytokinesis?

Answer 14

ANKLE1

Final checker

Question 15

What enzyme will we be discussing and why?

Answer 15

GEN1

most similar to most other organisms

Question 16

What family is GEN1 apart of?

Answer 16

FEN/EXO

Question 17

What is the FEN/EXO super-family?

Answer 17

structure selective nucleases

Question 18

What does GEN1 cleave?

Answer 18

3’ end to 4-way junction

Question 19

What type of GEN1 do we use to test upon its function?

Answer 19

CtGEN1-487 from furmophillic fungi

as GEN1 AGGREGATES

Question 20

How do we experimentally locate cleavages?

Answer 20

radioactively tag a strand of DNA on the junction and assay shows the cleaved strand
Run those products on a higher resolution sequencing gel.

Question 21

What does a higher resolution sequencing gel tell us?

Answer 21

determine to a nucleotide level where those cleavages occur .

Question 22

What are the strong cleavages?

Answer 22

1 nucleotide in the 3’ direction

Question 23

What do results look like if CtGEN1 binds discretley to DNA?

Answer 23

a slow migration on the gel

Question 24

What does CtGEN1 which binds discretely have?

Answer 24

high affinity for DNA junction

Question 25

How do we know GEN1 binds as a dimer?

Answer 25

We express as a fusion protein which increases the mass of the protein –>GEN1-GFP
Small form GEN1, big form GEN1-GFP fusion.
As we add more of the fusion form of GEN1 we now get 3 bands instead of 1
As it is a dimer, we can form 3 forms.

Question 26

What are the 3 forms of GEN1 we can add to the junction?

Answer 26

1. fastest = non fusion to non fusion
2. 1 fusion 1 non-fusion
3. fusion-fusion dimer species
   tells us that GEN1 is bound as a dimer