Week 5 L5: RuvC Flashcards
What is the function of RuvC?
the junction gets resolved back into 2 duplexes. Required for all methods of recombination.
What is the main player in the resolution of junction?
RuvC
dimeric nuclease, specific for the structure of the 4-way junction.
How many cleavage events occur in resolution?
2, in orderto separate 2 duplexes from the junction
What enzyme cleaves the four-way junction in bacteriophage infected bacteria?
T4 endonuclease VII
Where does the T4 endonuclease VII cleave?
2 cleavages symmetrically disposed about the junction. 2 nucleotides to the point of strand exchange.
What is the human version of RuvC?
GEN1
Are these enzymes basic or acidic?
BASIC
lots or Arg and Lys residues so it can bind to DNA covered in phosphate groups
Is RuvC dimeric or trimeric?
Dimeric
binds as a dimer (GEN1 too)
How many junction resolving activities are there in humans?
multiple
As this is a really important process and cant rely on just 1 activity
How are these resolution activities separated in humans?
spatially and temporally (different parts of the cell cycle)
What are the enzymes in G2/M phase in humans?
SLX1-SLX4-MUS81-EME1
What enzyme is present in anaphase in humans?
GEN1
Where is GEN1 present?
As it can only reach the nucleus when the nuclear membrane breaks down
What enzyme is present in cytokinesis?
ANKLE1
Final checker
What enzyme will we be discussing and why?
GEN1
most similar to most other organisms
What family is GEN1 apart of?
FEN/EXO
What is the FEN/EXO super-family?
structure selective nucleases
What does GEN1 cleave?
3’ end to 4-way junction
What type of GEN1 do we use to test upon its function?
CtGEN1-487 from furmophillic fungi
as GEN1 AGGREGATES
How do we experimentally locate cleavages?
radioactively tag a strand of DNA on the junction and assay shows the cleaved strand
Run those products on a higher resolution sequencing gel.
What does a higher resolution sequencing gel tell us?
determine to a nucleotide level where those cleavages occur .
What are the strong cleavages?
1 nucleotide in the 3’ direction
What do results look like if CtGEN1 binds discretley to DNA?
a slow migration on the gel
What does CtGEN1 which binds discretely have?
high affinity for DNA junction
How do we know GEN1 binds as a dimer?
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.
What are the 3 forms of GEN1 we can add to the junction?
- fastest = non fusion to non fusion
- 1 fusion 1 non-fusion
- fusion-fusion dimer species
tells us that GEN1 is bound as a dimer
