Sharma Neelam, Uhrig Mollie, Kwon Youngho, Sung Patrick, Wiese Claudia
bioRxiv. 2025 Sep 10:2025.09.10.675389. doi: 10.1101/2025.09.10.675389.
Homology-directed DNA repair (HDR) is critical for genome stability and tumor suppression. HDR is initiated by the RAD51 single-stranded (ss)DNA nucleoprotein filament which conducts the homology search and invades a homologous DNA template, creating a displacement-loop (D-loop). The RAD51 filament is assisted in these processes by several proteins. One such protein is RAD51-Associated-Protein 1 (RAD51AP1) which binds DNA and RNA and directly interacts with RAD51. Of note, RAD51AP1 overexpression is associated with poor prognosis in several different cancer types. Here, we show that RAD51AP1 activity is regulated by phosphorylation. RAD51AP1 bearing S277/282A mutations is more proficient in the stimulation of D-loop formation than wild type RAD51AP1 or phosphomimetic RAD51AP1-S277/282D. In EMSAs, RAD51AP1 with S277/282A mutations more avidly binds ssDNA, double-stranded (ds)DNA, and the nucleosome core particle than wild type RAD51AP1 or RAD51AP1-S277/282D. In cells, RAD51AP1-S277/282A confers no rescue of RAD51AP1 deficiency in toxicity tests and DNA replication assays. In contrast, RAD51AP1-S277/282D fully rescues RAD51AP1 deficiency. We provide evidence that RAD51AP1-S277 is a CDK2 target and propose a model in which RAD51AP1-S277/282 phosphorylation ensures RAD51AP1 flexibility for dynamic engagement in consecutive steps of the HDR reaction. Our results provide new mechanistic insights into RAD51AP1 regulation by a CDK.
同源重组介导的DNA修复（HDR）对于基因组稳定性和肿瘤抑制至关重要。HDR由RAD51单链（ss）DNA核蛋白丝启动，该丝进行同源性搜索并侵入同源DNA模板，形成置换环（D环）。几种蛋白质在这些过程中协助RAD51丝。其中一种蛋白质是RAD51相关蛋白1（RAD51AP1），它结合DNA和RNA并直接与RAD51相互作用。值得注意的是，RAD51AP1过表达与几种不同癌症类型的不良预后相关。在这里，我们表明RAD51AP1活性受磷酸化调节。携带S277/282A突变的RAD51AP1在刺激D环形成方面比野生型RAD51AP1或模拟磷酸化的RAD51AP1-S277/282D更有效。在电泳迁移率变动分析（EMSA）中，具有S277/282A突变的RAD51AP1比野生型RAD51AP1或RAD51AP1-S277/282D更 avidly 结合单链DNA、双链（ds）DNA和核小体核心颗粒。在细胞中，RAD51AP1-S277/282A在毒性试验和DNA复制试验中不能挽救RAD51AP1缺陷。相比之下，RAD51AP1-S277/282D完全挽救了RAD51AP1缺陷。我们提供证据表明RAD51AP1-S277是CDK2的靶标，并提出一个模型，其中RAD51AP1-S277/282磷酸化确保RAD51AP1的灵活性，以便在HDR反应的连续步骤中动态参与。我们的结果为CDK对RAD51AP1的调节提供了新的机制见解。
