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The SUMO modification pathway is involved in the BRCA1 response to genotoxic stress

Abstract

Mutations in BRCA1 are associated with a high risk of breast and ovarian cancer. BRCA1 participates in the DNA damage response and acts as a ubiquitin ligase. However, its regulation remains poorly understood. Here we report that BRCA1 is modified by small ubiquitin-like modifier (SUMO) in response to genotoxic stress, and co-localizes at sites of DNA damage with SUMO1, SUMO2/3 and the SUMO-conjugating enzyme Ubc9. PIAS SUMO E3 ligases co-localize with and modulate SUMO modification of BRCA1, and are required for BRCA1 ubiquitin ligase activity in cells. In vitro SUMO modification of the BRCA1/BARD1 heterodimer greatly increases its ligase activity, identifying it as a SUMO-regulated ubiquitin ligase (SRUbL). Further, PIAS SUMO ligases are required for complete accumulation of double-stranded DNA (dsDNA) damage-repair proteins subsequent to RNF8 accrual, and for proficient double-strand break repair. These data demonstrate that the SUMOylation pathway plays a significant role in mammalian DNA damage response.

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Figure 1: The SUMO-conjugation machinery locates to sites of DNA damage, and BRCA1 is modified by SUMO after genotoxic stress.
Figure 2: PIAS E3 SUMO ligases modulate BRCA1 SUMOylation.
Figure 3: PIAS E3 SUMO ligases influence accumulation of DNA damage-repair protein and are required for dsDNA break repair.
Figure 4: The SUMO pathway regulates BRCA1 ubiquitin ligase activity.

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Acknowledgements

We are grateful to V. De Laurenzi for the PIAS1 expression constructs, to R. Hay for His-SUMO1 and His-SUMO2 cells, to D. Durocher for anti-RNF168 antibody, to G. Stewart for discussions and S. Jackson for sharing results before publication. The work was supported by grants from Breast Cancer Campaign (to J.R.M., A.A., #SF06, and L.B., #06NovPHD13Morris), Cancer Research UK (to R.D., #C8820/A9494), the Medical Research Council (to E.S. and D.W., #6900577, and C.B.), the Richard Dimbleby Cancer Fund to King’s College London (to M.K. and T.N.) and Breakthrough Breast Cancer (to T.K). Multiphoton FLIM systems and acquisition/analysis software were built by S. Ameer-Beg, P. Barber and B. Vojnovic, with support from MRC Co-operative Group Grant G0100152 #56891 and UK Research Councils Basic Technology Research Programme Grant GR/R87901/01.

Author Contributions J.R.M. conceived and designed the study, generated reagents, performed experiments and wrote the paper. C.B. performed in vitro assays, confirmed and developed the initial concept, and generated reagents. M.K. optimised and performed FLIM measurements and analysis. R.D. and D.W. performed experiments and generated reagents. L.B. performed co-localisation observations, A.A. and L.P. generated reagents and Y.G. generated reagents and participated in discussions. T.K. undertook FLIM measurements. T.N. provided expertise and input into the design of the FLIM experiments, and E.S. provided advice and mentoring to J.R.M.

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Morris, J., Boutell, C., Keppler, M. et al. The SUMO modification pathway is involved in the BRCA1 response to genotoxic stress. Nature 462, 886–890 (2009). https://doi.org/10.1038/nature08593

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