Week 5 L6: Ct.GEN1

Question 1

How was the structure of CtGEN1 determined?

Answer 1

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

Question 2

What domain does a CtGEN1 monomer have?

Answer 2

chromodomain

Question 3

How does GEN1 bind to DNA?

Answer 3

monomer Gen1 binds to 2 arms of the 4 way junction (right angles).
Binds to the basic
Electrostatically attracted to Phospodiesteir groups.

Question 4

What regions does GEN1 have?

Answer 4

Electronegative acidic (lys, arg,) and electropositive basic (asp, glu)

Question 5

How does the GEN1 bind to correct position?

Answer 5

The alphahelixes at centre of structure are burring into the splitting at the junction.

Question 6

What part of the enzyme is acidic?

Answer 6

Active site

Question 7

How many acidic residues are there?

Answer 7

6
Asp or Glu
coordinate 2 metal ions

Question 8

How many metal ions are at the active site?

Answer 8

2

M1 & M2

Question 9

What is the role of the DNA in the active site?

Answer 9

Organise the active centre. Delivers the hydrolytic water molecule which hydrolyses the phosphodiester backbone.

Question 10

How do the 2 monomers interact?

Answer 10

dimer interface

Question 11

What interactions are there in the dimer interface?

Answer 11

3 alpha-helices from each monomer in intimate association baring a signif surface area.
Alpha 5, 4, 12 to 13, 4, 5

Question 12

What is the result of the dimer interface?

Answer 12

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.

Question 13

What arms are the sites of cleavage?

Answer 13

90 degree arms with metal ions

Question 14

Do the cleavages occur in one step?

Answer 14

NO, one cleavage and then the other

so it does not get left as a hemicleave junction

Question 15

What ensures a productive resolution event?

Answer 15

The 2nd cleavage is accelerated compared to the first.

Question 16

What event creates the duplex species?

Answer 16

ligation event

Question 17

How do you find a junction in the middle of DNA? (hypothesis)

Answer 17

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.

Question 18

Is this a set process in GEN1?

Answer 18

NO only the lecturer’s idea of how it could work

Question 19

What are the 3 steps in the Diffusion model?

Answer 19

Diffusion
Encounter
Dimer Assembly

Question 20

How do we explore this binding experimentally?

Answer 20

Optical trapping of DNA

C-trap apparatus

Question 21

How does optical trapping of DNA work?

Answer 21

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.

Question 22

What is the bead help in?

Answer 22

Laser trap

Question 23

How is the optical trapping of DNA contained?

Answer 23

microfluidic flow cell device

Question 24

What is added to GEN1 so it can be observed?

Answer 24

fluorescent tag

fluorescin.

Question 25

What graphs are produced from this experiment?

Answer 25

Kymographs | displacement against time

Question 26

How can you tell when the protein has found the junction from a kryograph?

Answer 26

The green line stabilises in the middle where the junction is. Stable manner of binding seen It can detach from unction

Question 27

What kind of diffusion is seen on a kryograph?

Answer 27

1D

Question 28

What are the resolution enzymes in G2/M phase?

Answer 28

SLX1, MUS81, EME1 organised by SLX4.

Question 29

What is SLX4?

Answer 29

tetrameric junction resolving enzyme

Question 30

What end of SLX4 does it bind the other enzymes?

Answer 30

C-terminus

Question 31

What are the nucleases in the SLX complex?

Answer 31

SLX1 & MUS81

Question 32

Is the SLX complex cleavage discrete?

Answer 32

YES

Question 33

What are the enzymes for resolution in cytokinesis?

Answer 33

ANKLE1/LEM3 | basically homologous

Question 34

Why is this enzyme complex important?

Answer 34

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

Question 35

What is the mammalian enzyme for cytokinesis resolution?

Answer 35

ANKLE1

Question 36

What is the C. elegans enzyme for cytokinesis?

Answer 36

LEM3

Question 37

Where is LEM3 located?

Answer 37

During anaphase it can be seen localised at the mid-body.

Question 38

What does LEM3 process when cells are separating?

Answer 38

DNA ultra fine bridges are processed by this nuclease

Question 39

What kind of enzymes are ANKLE1 and LEM?

Answer 39

NUCLEASES

Question 40

Why do human ANKLE1 have a lower specificity?

Answer 40

so it can cleave a wide variety of branched DNA species. Process any junction that remains. Allowing the cells to divide.