Fondazione IFOM, Istituto FIRC di Oncologia Molecolare, Milan, Italy.
PLoS Genet. 2010 Nov 11;6(11):e1001205. doi: 10.1371/journal.pgen.1001205.
Damage tolerance mechanisms mediating damage-bypass and gap-filling are crucial for genome integrity. A major damage tolerance pathway involves recombination and is referred to as template switch. Template switch intermediates were visualized by 2D gel electrophoresis in the proximity of replication forks as X-shaped structures involving sister chromatid junctions. The homologous recombination factor Rad51 is required for the formation/stabilization of these intermediates, but its mode of action remains to be investigated. By using a combination of genetic and physical approaches, we show that the homologous recombination factors Rad55 and Rad57, but not Rad59, are required for the formation of template switch intermediates. The replication-proficient but recombination-defective rfa1-t11 mutant is normal in triggering a checkpoint response following DNA damage but is impaired in X-structure formation. The Exo1 nuclease also has stimulatory roles in this process. The checkpoint kinase, Rad53, is required for X-molecule formation and phosphorylates Rad55 robustly in response to DNA damage. Although Rad55 phosphorylation is thought to activate recombinational repair under conditions of genotoxic stress, we find that Rad55 phosphomutants do not affect the efficiency of X-molecule formation. We also examined the DNA polymerase implicated in the DNA synthesis step of template switch. Deficiencies in translesion synthesis polymerases do not affect X-molecule formation, whereas DNA polymerase δ, required also for bulk DNA synthesis, plays an important role. Our data indicate that a subset of homologous recombination factors, together with DNA polymerase δ, promote the formation of template switch intermediates that are then preferentially dissolved by the action of the Sgs1 helicase in association with the Top3 topoisomerase rather than resolved by Holliday Junction nucleases. Our results allow us to propose the choreography through which different players contribute to template switch in response to DNA damage and to distinguish this process from other recombination-mediated processes promoting DNA repair.
介导损伤绕过和间隙填充的损伤耐受机制对于基因组完整性至关重要。一个主要的损伤耐受途径涉及重组,被称为模板转换。通过二维凝胶电泳,在复制叉附近观察到模板转换中间体,表现为涉及姐妹染色单体连接的 X 形结构。同源重组因子 Rad51 是形成/稳定这些中间体所必需的,但它的作用模式仍有待研究。通过使用遗传和物理方法的组合,我们表明同源重组因子 Rad55 和 Rad57,但不是 Rad59,对于模板转换中间体的形成是必需的。复制有效的但重组缺陷的 rfa1-t11 突变体在 DNA 损伤后触发检查点反应是正常的,但在 X 结构形成中受损。外切核酸酶 Exo1 在此过程中也具有刺激作用。检查点激酶 Rad53 对于 X 分子的形成是必需的,并在 DNA 损伤后强烈磷酸化 Rad55。虽然 Rad55 磷酸化被认为在遗传毒性应激条件下激活重组修复,但我们发现 Rad55 磷酸突变体不会影响 X 分子形成的效率。我们还检查了参与模板转换 DNA 合成步骤的 DNA 聚合酶。跨损伤合成聚合酶的缺陷不会影响 X 分子的形成,而 DNA 聚合酶 δ,也需要进行大量的 DNA 合成,起着重要的作用。我们的数据表明,一组同源重组因子与 DNA 聚合酶 δ 一起,促进模板转换中间体的形成,然后这些中间体通过 Sgs1 解旋酶与 Top3 拓扑异构酶的关联而优先溶解,而不是通过 Holliday 连接酶进行解决。我们的结果使我们能够提出不同的参与者如何响应 DNA 损伤贡献于模板转换的编排,并将此过程与其他促进 DNA 修复的重组介导过程区分开来。