C62
DNA damage induces SUMO modification of BRCA1
Jo Morris1, Chris Boutell2, Melanie Keppler3, Amin Alamshah1, Laura Butler1, Laurent Pangon1, Tony Ng3, Ellen Solomon1
1Dept Medical and Molecular Genetics, King's College London, London, UK, 2Institute of Virology, University of Glasgow, Glasgow, UK, 3Randall Division of Cell and Molecular Biopysics, King's College London, London, UK
The BRCA1 breast and ovarian cancer predisposition protein is thought to function predominantly in the nucleus to regulate cell cycle progression, DNA repair/recombination and gene transcription. Currently the regulation of these functions and any cross-talk between them is poorly understood.
We have investigated potential regulation of BRCA1 by post-transitional modification with the Small Ubiquitin like Modifier. In our initial observations we noted that SUMO isoforms and BRCA1 co-localised following DNA damage. We showed modification of BRCA1 itself by nickel purification of hexahistidine tagged SUMO conjugates from cells. BRCA1 was purified following DNA replication block and collapse and cellular stress. These data also indicated preferential modification of BRCA1 by SUMO-2/3 over SUMO-1 isoforms.
We demonstrate that the N-terminal region of BRCA1 is able to interact with the SUMO E2 conjugated enzyme, Ubc9 and be modified in vitro by SUMO conjugation components. In cells we monitored BRCA1 SUMOylation via Fluorescence Lifetime Imaging Microscopy (FLIM), where interaction was dependent on the C-terminal glycine of SUMO and increased by DNA damage. Mutation of a SUMO modification consensus sequence at the N-terminus inhibited SUMO:BRCA1 interaction suggesting it is the site of conjugation. Loss of this site altered BRCA1 degradation regulated by the proteasome and nuclear export directed by interaction with the CRM1-nuclear export protein. Hence DNA damage results in SUMO conjugation of BRCA1 to regulate BRCA1 protein stability and subcellular location.
