Department of Biochemistry & Molecular Biophysics, Columbia University, New York, NY, USA.
Department of Biochemistry and Structural Biology, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA.
EMBO J. 2020 Oct 15;39(20):e105705. doi: 10.15252/embj.2020105705. Epub 2020 Aug 13.
Rad54 and Rdh54 are closely related ATP-dependent motor proteins that participate in homologous recombination (HR). During HR, these enzymes functionally interact with the Rad51 presynaptic complex (PSC). Despite their importance, we know little about how they are organized within the PSC, or how their organization affects PSC function. Here, we use single-molecule optical microscopy and genetic analysis of chimeric protein constructs to evaluate the binding distributions of Rad54 and Rdh54 within the PSC. We find that Rad54 and Rdh54 have distinct binding sites within the PSC, which allow these proteins to act cooperatively as DNA sequences are aligned during homology search. Our data also reveal that Rad54 must bind to a specific location within the PSC, whereas Rdh54 retains its function in the repair of MMS-induced DNA damage even when recruited to the incorrect location. These findings support a model in which the relative binding sites of Rad54 and Rdh54 help to define their functions during mitotic HR.
Rad54 和 Rdh54 是密切相关的 ATP 依赖性运动蛋白，它们参与同源重组 (HR)。在 HR 过程中，这些酶与 Rad51 前突触复合物 (PSC) 进行功能相互作用。尽管它们很重要，但我们对它们在 PSC 中的组织方式知之甚少，也不知道它们的组织方式如何影响 PSC 的功能。在这里，我们使用单分子光学显微镜和嵌合蛋白构建体的遗传分析来评估 Rad54 和 Rdh54 在 PSC 中的结合分布。我们发现 Rad54 和 Rdh54 在 PSC 内具有不同的结合位点，这允许这些蛋白在同源性搜索过程中 DNA 序列对齐时协同作用。我们的数据还表明，Rad54 必须结合到 PSC 内的特定位置，而 Rdh54 即使被招募到错误的位置，也能保持其在 MMS 诱导的 DNA 损伤修复中的功能。这些发现支持了这样一种模型，即 Rad54 和 Rdh54 的相对结合位点有助于在有丝分裂 HR 期间定义它们的功能。
