Ubiquitation

Question 1

Describe the conjugation of a target protein by ubiquitin

Answer 1

Added onto a lysine residue by 3 enzymes- requires ATP

E1 is uniquitinated➡️ transfers Ub to E2➡️ E3 transfers Ub to the substrate

Question 2

How many different ubiquitin chains are there?

Answer 2

8
Eg.
K-63 linked 
K-48 linked
Mono-Ub

Question 3

What is the outcome of k-48 linked ubiquitination?

Answer 3

Degradation by a proteosome

Question 4

Describe ubiquitin writers in response to DNA DSB

Answer 4

E3 ubiquitin ligases
Eg. RNF8 and RNF168
MRN➡️ MDC1➡️ RNF8➡️ E2 Ub-ates H1K48 and K63, leading to the eviction and degradation of the chromatin bound factor JMJD2A, revealing the H4K20Me3 mark.
RNF8 activity leads to the recruitment of another E3 ubiquitin ligase RNF168, that has UIM - it reads the H1K63.
RNF8 and RNF168 mono-ubiquitinates at K15/13 on H2A in the nucleosome adjacent to the DSB and create K63-linked Ub chains on H1
This then recruits 53BP1 and BRCA1.

Question 5

How do RNF8 and RNF168 affect the division point of DSB repair?

Answer 5

53BP1 reads the H4K20Me3 and the H2A K13/15Ub
And prevents the resection of the DNA thus promoting NHEJ. 53BP1 binds RIF1 that blocks BRCA1 to prevent DNA resection.
In S-phase - RAP80 reads the K63Ub on H1. Recruits Abraxas, BRCA1 and thus ATM.
BRCA1 and CtIP recruitment evicts 53BP1 allowing resection of the DNA and therefore HR.

Question 6

Iso-forms of SUMO

Answer 6

SUMO1 can mono-SUMOylate

SUMO2/3 can multi-monoSUMOylate and poly-SUMOylate

Question 7

Describe SUMO conjugation

Answer 7

SUMOylation occurs at large hydrophobic regions before lysine residues and after negative charged region.
SIM is a SUMO interacting motif.
RNF4 can recognise both SUMO and Ub

Question 8

Name DUBs

Answer 8

USP3 - inhibits recruitment of RNF168, 53BP1 and RAP80
USP44 - overexpression prevents 53BP1 forming
POH1 -limits 53BP1 by recognising K63-Ub-linkages
SENP7 - deSUMOylates to promote chromatin relaxation and HR

Question 9

What is the role of DUBs?

Answer 9

Control the degree of ubiquitin conjugation
Have the potential to modulate DNA repair
SUMO-processing is in part ache iced by RNF4 and partly unidentified

Question 10

What is SUMO?

Answer 10

Small ubiquitin-like modifier