Dubois Jean-Christophe, Yates Maïlyn, Gaudreau-Lapierre Antoine, Clément Geneviève, Cappadocia Laurent, Gaudreau Luc, Zou Lee, Maréchal Alexandre
Department of Biology, Université de Sherbrooke, Sherbrooke, QC J1K 2R1, Canada.
Structural Biology Program, Sloan Kettering Institute, New York, NY 10021, USA.
Nucleic Acids Res. 2017 Sep 6;45(15):8859-8872. 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.
RPA 包被的单链 DNA（RPA-ssDNA）是一种由 DNA 损伤诱导产生的核蛋白结构，可促进 ATR 激活和同源重组（HR）。DNA 损伤后，RPA 会发生过度磷酸化和泛素化。PRP19 和 RFWD3 介导的 RPA 泛素化促进 ATR 激活和 HR，但 DNA 损伤如何刺激这一过程仍不清楚。在此，我们发现 RFWD3 持续结合 RPA，而 PRP19 在 DNA 损伤后识别 RPA。RPA 对 PRP19 的招募依赖于 PIKK 介导的 RPA 磷酸化以及 PRP19 中的一个带正电荷的口袋。缺乏磷酸化位点的 RPA32 突变体无法招募 PRP19 并支持 RPA 泛素化。无法结合 RPA 或缺乏泛素连接酶活性的 PRP19 突变体也无法支持 RPA 泛素化和 HR。这些结果表明，RPA 磷酸化增强了 PRP19 向 RPA-ssDNA 的招募，并通过一个需要 PRP19 和 RFWD3 的过程刺激 RPA 泛素化，从而触发一个促进 ATR 激活和 HR 的磷酸化-泛素化回路。
