Two Redundant Ubiquitin-dependent Pathways of BRCA1 Localization to DNA Damage Sites

The tumor suppressor BRCA1 accumulates at sites of DNA damage in a ubiquitin-dependent manner, and coordinates DSB repair. The phosphopeptide-binding activity of the BRCA1 C-terminal (BRCT) domains is essential for ubiquitin-dependent recruitment and it is through these domains that BRCA1 indirectly interacts with RAP80, a ubiquitin-interacting protein. RAP80 targets BRCA1 to DSBs but whether or not RAP80 is the sole targeting subunit of the BRCA1 complex is a subject of debate. In my thesis, I generated somatic RAP80-/- cell lines and found that RAP80 is dispensable for the initial recruitment of BRCA1 to DSBs. I observed that the isolated tandem BRCT domain absolutely requires RAP80 to localize at DSBs, but the full-length protein does not. I found that the BRCA1 RING domain acts redundantly to promote BRCA1 recruitment to DNA damage sites. I show that that RNF8 E3 ligase acts upstream of both the RAP80- and RING-dependent activities whereas RNF168 acts uniquely upstream of the RING domain. BRCA1 RING mutations that do not impact BARD1 interaction, such as the E2-binding deficient I26A mutation, renders BRCA1 unable to accumulate at DNA damage sites in the absence of RAP80. Cells that combine BRCA1 I26A and mutations that disable the RAP80-BRCA1 interaction are hypersensitive to PARP inhibition and are unable to form RAD51 foci. Our results suggest that in the absence of RAP80, the BRCA1 E3 ligase activity is necessary for recognition of histone H2A Lys13/Lys15 ubiquitylation by BARD1 although I cannot rule out the possibility that the BRCA1 RING facilitates ubiquitylated nucleosome recognition in other ways.

Department dc:contributor.department

Molecular Genetics

Year dc:date.issued

2022

Author dc:creator

• Sherker, Alana Rose

Advisor dc:contributor.advisor

• Durocher, Daniel

• BARD1
• BRCA1
• DNA damage
• RAP80
• ubiquitin

Statement dc:rights

• Attribution 4.0 International

Licence dc:rights.uri

• http://creativecommons.org/licenses/by/4.0/

Handle dc:identifier.uri

http://hdl.handle.net/1807/123588

OAI identifier oai:identifier

oai:utoronto.scholaris.ca:1807/123588