Repair of DNA double-stranded breaks by homologous recombination (HR) is dependent on DNA end resection and on post-translational modification of repair factors. In budding yeast, single-stranded DNA is coated by replication protein A (RPA) following DNA end resection, and DNA–RPA complexes are then SUMO-modified by the E3 ligase Siz2 to promote repair. Here, we show using enzymatic assays that DNA duplexes containing 3' single-stranded DNA overhangs increase the rate of RPA SUMO modification by Siz2. The SAP domain of Siz2 binds DNA duplexes and makes a key contribution to this process as highlighted by models and a crystal structure of Siz2 and by assays performed using protein mutants. Enzymatic assays performed using DNA that can accommodate multiple RPA proteins suggest a model in which the SUMO-RPA signal is amplified by successive rounds of Siz2-dependent SUMO modification of RPA and dissociation of SUMO-RPA at the junction between single- and double-stranded DNA. Our results provide insights on how DNA architecture scaffolds a substrate and E3 ligase to promote SUMO modification in the context of DNA repair.

中文翻译：

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DNA不对称促进单链DNA结合蛋白RPA的SUMO修饰

通过同源重组 (HR) 修复 DNA 双链断裂取决于 DNA 末端切除和修复因子的翻译后修饰。在出芽酵母中，单链 DNA 在 DNA 末端切除后被复制蛋白 A (RPA) 包被，然后 DNA-RPA 复合物通过 E3 连接酶 Siz2 进行 SUMO 修饰以促进修复。在这里，我们使用酶分析表明，含有 3' 单链 DNA 悬垂的 DNA 双链体增加了 Siz2 对 RPA SUMO 修饰的速率。Siz2 的 SAP 结构域结合 DNA 双链体，并对这一过程做出了关键贡献，正如模型和 Siz2 的晶体结构以及使用蛋白质突变体进行的分析所强调的那样。使用可容纳多种 RPA 蛋白的 DNA 进行的酶促测定表明，在这种模型中，SUMO-RPA 信号通过 RPA 的连续多轮 Siz2 依赖性 SUMO 修饰和单链和双链连接处的 SUMO-RPA 解离来放大脱氧核糖核酸。我们的研究结果提供了有关 DNA 结构如何支撑底物和 E3 连接酶以在 DNA 修复背景下促进 SUMO 修饰的见解。