RNF8 E3 Ubiquitin Ligase Stimulates Ubc13 E2 Conjugating Activity That Is Essential for DNA Double Strand Break Signaling and BRCA1 Tumor Suppressor Recruitment.
J Biol Chem
; 291(18): 9396-410, 2016 Apr 29.
Article
in En
| MEDLINE
| ID: mdl-26903517
ABSTRACT
DNA double strand break (DSB) responses depend on the sequential actions of the E3 ubiquitin ligases RNF8 and RNF168 plus E2 ubiquitin-conjugating enzyme Ubc13 to specifically generate histone Lys-63-linked ubiquitin chains in DSB signaling. Here, we defined the activated RNF8-Ubc13â¼ubiquitin complex by x-ray crystallography and its functional solution conformations by x-ray scattering, as tested by separation-of-function mutations imaged in cells by immunofluorescence. The collective results show that the RING E3 RNF8 targets E2 Ubc13 to DSB sites and plays a critical role in damage signaling by stimulating polyubiquitination through modulating conformations of ubiquitin covalently linked to the Ubc13 active site. Structure-guided separation-of-function mutations show that the RNF8 E2 stimulating activity is essential for DSB signaling in mammalian cells and is necessary for downstream recruitment of 53BP1 and BRCA1. Chromatin-targeted RNF168 rescues 53BP1 recruitment involved in non-homologous end joining but not BRCA1 recruitment for homologous recombination. These findings suggest an allosteric approach to targeting the ubiquitin-docking cleft at the E2-E3 interface for possible interventions in cancer and chronic inflammation, and moreover, they establish an independent RNF8 role in BRCA1 recruitment.
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Signal Transduction
/
Tumor Suppressor Proteins
/
Ubiquitin-Conjugating Enzymes
/
Ubiquitin-Protein Ligases
/
DNA Breaks, Double-Stranded
/
Ubiquitination
Limits:
Animals
Language:
En
Journal:
J Biol Chem
Year:
2016
Type:
Article
Affiliation country:
Canada