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 H1 leads to the eviction and degradation of the chromatin bound factor JMJD2A
RNF8 activity leads to the recruitment of another E3 ubiquitin ligase RNF168, that has ubiquitin interaction motifs (UIM)- it reads the uniquitinated histone
RNF8 and RNF168 mono-ubiquitinates H2A in the nucleosome adjacent to the DSB and create K63-linked Ub chains on H1

Question 5

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

Answer 5

53BP1 reads the H4K20Me3 and the H2A-Ub- somehow encouraged by the K63-linked Ub chain
And prevents the resection of the DNA thus promoting NHEJ in G1
53BP1 binds RIF1 that blocks BRCA1 to prevent DNA resection
In late S-phase, G2- RAP80 reads the K63-linked Ub on H1➡️ Abraxas➡️ BRCA1➡️ ATM
BRCA1 and CtIP recruitment evicts 53BP1 allowing resection of the DNA and therefore HR

Question 6

Name two SUMO E3 ligases or ‘writers’

Answer 6

PIAS1 and PIAS4 (Siz2 in yeast)

Question 7

Describe SUMO conjugation

Answer 7

Writers- SUMO E3 ligases
Readers- proteins that contain SUMO Interaction Motifs (SIMs)
SUMO conjugation to DNA repair proteins induces higher order complexes through SUMO:SIM interactions
Promotes the clearance of DNS repair proteins via the activity of a SUMO targeted ubiquitin ligase, RNF4, which has SIMs

Question 8

Name some ‘editors’ of protein modification associated with H2A-Ub deubiquitination

Answer 8

USP16
BAP1
USP3- inhibits recruitment of RNF168, 53BP1 and RAP80
USP44- overexpression prevents 53BP1 forming
BRCC36 and POH1 are de-ubiquitinating enzymes that are specific to K63-Ub-linkages

Question 9

What is the role of editors or de-ubiquitination enzymes?

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