Mammalian SUMO E3-ligases PIAS1 and PIAS4 promote responses to DNA double-strand breaks.
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 repair. Attachment of small ubiquitin-related modifier (SUMO) to target proteins controls diverse cellular functions. 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 damage. 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.
Galanty
Yaron
Y
The Wellcome Trust and Cancer Research UK Gurdon Institute, and Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QN, UK.
Belotserkovskaya
Rimma
R
Coates
Julia
J
Polo
Sophie
S
Miller
Kyle M
KM
Jackson
Stephen P
SP
D000818:Animals; D019313:BRCA1 Protein / Q000378:metabolism; D002460:Cell Line; D045744:Cell Line, Tumor; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D036681:Fluorescence Recovery After Photobleaching; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D008954:Models, Biological; D010766:Phosphorylation; D050828:Protein Inhibitors of Activated STAT / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D051716:Replication Protein A / Q000378:metabolism; D025841:Small Ubiquitin-Related Modifier Proteins / Q000235:genetics / Q000378:metabolism*; D044763:Ubiquitin-Conjugating Enzymes / Q000235:genetics / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism; D054875:Ubiquitination; D000098843:Ubiquitin-Conjugating Enzyme UBC9
D019313:BRCA1 Protein; D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D050828:Protein Inhibitors of Activated STAT; D051716:Replication Protein A; D025841:Small Ubiquitin-Related Modifier Proteins; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; D000098843:Ubiquitin-Conjugating Enzyme UBC9