Week 5 L6: Ct.GEN1 Flashcards
How was the structure of CtGEN1 determined?
CtGEN1 is the pure form
crystallisation solve structure by X-ray by X-ray diffraction.
GEN1 with Mg2+ will activate the enzyme.
Will receive crystal’s with a complex of resolution with the product cleavage
What domain does a CtGEN1 monomer have?
chromodomain
How does GEN1 bind to DNA?
monomer Gen1 binds to 2 arms of the 4 way junction (right angles).
Binds to the basic
Electrostatically attracted to Phospodiesteir groups.
What regions does GEN1 have?
Electronegative acidic (lys, arg,) and electropositive basic (asp, glu)
How does the GEN1 bind to correct position?
The alphahelixes at centre of structure are burring into the splitting at the junction.
What part of the enzyme is acidic?
Active site
How many acidic residues are there?
6
Asp or Glu
coordinate 2 metal ions
How many metal ions are at the active site?
2
M1 & M2
What is the role of the DNA in the active site?
Organise the active centre. Delivers the hydrolytic water molecule which hydrolyses the phosphodiester backbone.
How do the 2 monomers interact?
dimer interface
What interactions are there in the dimer interface?
3 alpha-helices from each monomer in intimate association baring a signif surface area.
Alpha 5, 4, 12 to 13, 4, 5
What is the result of the dimer interface?
juxed opposed position
Coaxial alignment between 2 of the arms & other 2 arms come out at 90 degrees to that axis and at 90 degrees from each other.
What arms are the sites of cleavage?
90 degree arms with metal ions
Do the cleavages occur in one step?
NO, one cleavage and then the other
so it does not get left as a hemicleave junction
What ensures a productive resolution event?
The 2nd cleavage is accelerated compared to the first.
What event creates the duplex species?
ligation event
How do you find a junction in the middle of DNA? (hypothesis)
DIFFUSION MODEL.
Protein bind remotely in DNA. Bind as monomer to one arm of the junction then slide along DNA until it encounters a junction and is bound to 2 of the arms.
Might have a 2nd monomer sliding in from the other direction.
Assemble the dimeric complex at junction.
Is this a set process in GEN1?
NO only the lecturer’s idea of how it could work
What are the 3 steps in the Diffusion model?
Diffusion
Encounter
Dimer Assembly
How do we explore this binding experimentally?
Optical trapping of DNA
C-trap apparatus
How does optical trapping of DNA work?
Have a junction
The DNA on either size is attached to a bead and the bead is held in an optical trap.
Apply stretching so the DNA is approximately linear.
Observe Fluorescent GEN1 to hopefully watch it bind to DNA and can track it.
Can be viewed under the microfluidic flow cell microscope.
What is the bead help in?
Laser trap
How is the optical trapping of DNA contained?
microfluidic flow cell device
What is added to GEN1 so it can be observed?
fluorescent tag
fluorescin.
What graphs are produced from this experiment?
Kymographs
displacement against time
How can you tell when the protein has found the junction from a kryograph?
The green line stabilises in the middle where the junction is.
Stable manner of binding seen
It can detach from unction
What kind of diffusion is seen on a kryograph?
1D
What are the resolution enzymes in G2/M phase?
SLX1, MUS81, EME1 organised by SLX4.
What is SLX4?
tetrameric junction resolving enzyme
What end of SLX4 does it bind the other enzymes?
C-terminus
What are the nucleases in the SLX complex?
SLX1 & MUS81
Is the SLX complex cleavage discrete?
YES
What are the enzymes for resolution in cytokinesis?
ANKLE1/LEM3
basically homologous
Why is this enzyme complex important?
As it is the last step in the process, if any junction fails to be cleaved or processed then this enzyme can catch it.
Important as cell division cannot occur if there are any connections left between chromosomes.
get rid of residual junctions
What is the mammalian enzyme for cytokinesis resolution?
ANKLE1
What is the C. elegans enzyme for cytokinesis?
LEM3
Where is LEM3 located?
During anaphase it can be seen localised at the mid-body.
What does LEM3 process when cells are separating?
DNA ultra fine bridges are processed by this nuclease
What kind of enzymes are ANKLE1 and LEM?
NUCLEASES
Why do human ANKLE1 have a lower specificity?
so it can cleave a wide variety of branched DNA species. Process any junction that remains.
Allowing the cells to divide.
