A phosphorylation-and-ubiquitylation circuitry driving ATR activation and homologous recombination

• Published in: Nucleic acids research 2017-09, Vol.45 (15), p.8859-8872
• Main Authors: Dubois, Jean-Christophe, Yates, Maïlyn, Gaudreau-Lapierre, Antoine, Clément, Geneviève, Cappadocia, Laurent, Gaudreau, Luc, Zou, Lee, Maréchal, Alexandre
• Format: Article
• Language: English
• Subjects: Ataxia Telangiectasia Mutated Proteins - genetics; Ataxia Telangiectasia Mutated Proteins - metabolism; Cell Line, Tumor; DNA Damage; DNA Repair; DNA Repair Enzymes - chemistry; DNA Repair Enzymes - genetics; DNA Repair Enzymes - metabolism; DNA Replication; DNA, Single-Stranded - genetics; DNA, Single-Stranded - metabolism; Gene Expression Regulation; Genome Integrity, Repair and; HEK293 Cells; HeLa Cells; Homologous Recombination; Humans; Nuclear Proteins - chemistry; Nuclear Proteins - genetics; Nuclear Proteins - metabolism; Osteoblasts - cytology; Osteoblasts - metabolism; Phosphorylation; Replication Protein A - genetics; Replication Protein A - metabolism; RNA Splicing Factors - chemistry; RNA Splicing Factors - genetics; RNA Splicing Factors - metabolism; Signal Transduction; Ubiquitin-Protein Ligases - genetics; Ubiquitin-Protein Ligases - metabolism; Ubiquitination
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gkx571

RPA-coated single-stranded DNA (RPA-ssDNA), a nucleoprotein structure induced by DNA damage, promotes ATR activation and homologous recombination (HR). RPA is hyper-phosphorylated and ubiquitylated after DNA damage. The ubiquitylation of RPA by PRP19 and RFWD3 facilitates ATR activation and HR, but how it is stimulated by DNA damage is still unclear. Here, we show that RFWD3 binds RPA constitutively, whereas PRP19 recognizes RPA after DNA damage. The recruitment of PRP19 by RPA depends on PIKK-mediated RPA phosphorylation and a positively charged pocket in PRP19. An RPA32 mutant lacking phosphorylation sites fails to recruit PRP19 and support RPA ubiquitylation. PRP19 mutants unable to bind RPA or lacking ubiquitin ligase activity also fail to support RPA ubiquitylation and HR. These results suggest that RPA phosphorylation enhances the recruitment of PRP19 to RPA-ssDNA and stimulates RPA ubiquitylation through a process requiring both PRP19 and RFWD3, thereby triggering a phosphorylation-ubiquitylation circuitry that promotes ATR activation and HR.