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Mammalian SUMO E3-ligases PIAS1 and PIAS4 promote responses to DNA double-strand breaks

Abstract

DNA double-strand breaks (DSBs) are highly cytotoxic lesions that are generated by ionizing radiation and various DNA-damaging chemicals. Following DSB formation, cells activate the DNA-damage response (DDR) protein kinases ATM, ATR and DNA-PK (also known as PRKDC). These then trigger histone H2AX (also known as H2AFX) phosphorylation and the accumulation of proteins such as MDC1, 53BP1 (also known as TP53BP1), BRCA1, CtIP (also known as RBBP8), RNF8 and RNF168/RIDDLIN into ionizing radiation-induced foci (IRIF) that amplify DSB signalling and promote DSB repair1,2. Attachment of small ubiquitin-related modifier (SUMO) to target proteins controls diverse cellular functions3,4,5,6. Here, we show that SUMO1, SUMO2 and SUMO3 accumulate at DSB sites in mammalian cells, with SUMO1 and SUMO2/3 accrual requiring the E3 ligase enzymes PIAS4 and PIAS1. We also establish that PIAS1 and PIAS4 are recruited to damage sites via mechanisms requiring their SAP domains, and are needed for the productive association of 53BP1, BRCA1 and RNF168 with such regions. Furthermore, we show that PIAS1 and PIAS4 promote DSB repair and confer ionizing radiation resistance. Finally, we establish that PIAS1 and PIAS4 are required for effective ubiquitin-adduct formation mediated by RNF8, RNF168 and BRCA1 at sites of DNA damage7,8,9,10,11. These findings thus identify PIAS1 and PIAS4 as components of the DDR and reveal how protein recruitment to DSB sites is controlled by coordinated SUMOylation and ubiquitylation.

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Figure 1: SUMOs and UBC9 accumulate at DNA-damage sites by mechanisms requiring MDC1, 53BP1 and BRCA1.
Figure 2: PIAS1 and PIAS4 are recruited to DNA-damage sites and mediate 53BP1 recruitment and SUMOylation.
Figure 3: PIAS1 and PIAS4 promote BRCA1 accumulation and SUMOylation, RPA phosphorylation, and DSB repair.
Figure 4: Linkage between PIAS1/4 and RNF8/168.

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Acknowledgements

We thank S.P.J. lab members for support, in particular J. Harrigan, P. Huertas, S. Gravel, K. Dry and R. Chapman. We also thank C. Lukas for U2OS cells expressing GFP–BRCA1/Flag–BARD1, D. Durocher and G. Stewart for hTERT RIDDLE syndrome fibroblasts complemented with vector or HA–RNF168 and RNF168 antibody, T. Halazonetis for RNF8 antibody, R. Baer for the Flag–BARD1 construct, P. Harkin for the HA–BRCA1 construct, K. Iwabuchi for HA-tagged, full length, N, C, CΔBRCT and BRCT 53BP1 constructs, R. Walker for help with FACS, and J. R. Morris for sharing results before publication. Research in the S.P.J. lab is supported by grants from Cancer Research UK and the European Union (Integrated Project DNA repair, LSHG-CT-2005-512113, and Genomic Instability in Cancer and Precancer, HEALTH-F2-2007-201630).

Author Contributions R.B. cloned the PIAS cDNAs, tested the original siRNA efficiencies and provided help with processing of laser experiments. J.C. intensively helped with cell survival, homologous recombination and NHEJ experiments and provided support with tissue culture maintenance and stable-cell-line generation. S.P. set up the laser system in the laboratory and helped perform and analyse the FRAP experiments. K.M.M. provided the initial results on 53BP1 IRIF in PIAS4-depleted cells and constructed siRNA-resistant RFP–PIAS4. Y.G. initiated the project, led the teamwork and performed all other experiments described in the manuscript. Y.G. and S.P.J. conceived the study and wrote the paper. All authors discussed and commented on the manuscript.

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Correspondence to Stephen P. Jackson.

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Galanty, Y., Belotserkovskaya, R., Coates, J. et al. Mammalian SUMO E3-ligases PIAS1 and PIAS4 promote responses to DNA double-strand breaks. Nature 462, 935–939 (2009). https://doi.org/10.1038/nature08657

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