Lahiri Sudipta, Hamilton George, Moore Gemma, Goehring Liana, Huang Tony T, Jensen Ryan B, Rothenberg Eli
Department of Biochemistry and Molecular Pharmacology, New York University Grossman School of Medicine, New York, NY, USA.
Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT, USA.
Nature. 2025 Apr;640(8060):1103-1111. doi: 10.1038/s41586-025-08749-x. Epub 2025 Mar 26.
The tumour-suppressor protein BRCA2 has a central role in homology-directed DNA repair by enhancing the formation of RAD51 filaments on resected single-stranded DNA generated at double-stranded DNA breaks and stimulating RAD51 activity. Individuals with BRCA2 mutations are predisposed to cancer; however, BRCA2-deficient tumours are often responsive to targeted therapy with PARP inhibitors (PARPi). The mechanism by which BRCA2 deficiency renders cells sensitive to PARPi but with minimal toxicity in cells heterozygous for BRCA2 mutations remains unclear. Here we identify a previously unknown role of BRCA2 that is directly linked to the effect of PARP1 inhibition. Using biochemical and single-molecule approaches, we demonstrate that PARPi-mediated PARP1 retention on a resected DNA substrate interferes with RAD51 filament stability and impairs RAD51-mediated DNA strand exchange. Full-length BRCA2 protects RAD51 filaments and counteracts the instability conferred by PARPi-mediated retention by preventing the binding of PARP1 to DNA. Extending these findings to a cellular context, we use quantitative single-molecule localization microscopy to show that BRCA2 prevents PARPi-induced PARP1 retention at homologous-recombination repair sites. By contrast, BRCA2-deficient cells exhibit increased PARP1 retention at these lesions in response to PARPi. These results provide mechanistic insights into the role of BRCA2 in maintaining RAD51 stability and protecting homologous-recombination repair sites by mitigating PARPi-mediated PARP1 retention.
肿瘤抑制蛋白BRCA2在同源性定向DNA修复中发挥核心作用，它通过增强在双链DNA断裂处产生的经切除的单链DNA上RAD51丝的形成，并刺激RAD51活性来实现这一作用。携带BRCA2突变的个体易患癌症；然而，BRCA2缺陷型肿瘤通常对PARP抑制剂（PARPi）靶向治疗有反应。BRCA2缺陷使细胞对PARPi敏感，但对BRCA2突变杂合细胞毒性极小，其机制尚不清楚。在此，我们确定了BRCA2一个此前未知的作用，该作用与PARP1抑制的效果直接相关。利用生化和单分子方法，我们证明PARPi介导的PARP1在经切除的DNA底物上的滞留会干扰RAD51丝的稳定性，并损害RAD51介导的DNA链交换。全长BRCA2保护RAD51丝，并通过阻止PARP1与DNA结合来抵消PARPi介导的滞留所带来的不稳定性。将这些发现扩展到细胞环境中，我们使用定量单分子定位显微镜来表明BRCA2可防止PARPi诱导的PARP1在同源重组修复位点的滞留。相比之下，BRCA2缺陷型细胞在PARPi作用下，这些损伤处的PARP1滞留增加。这些结果为BRCA2在维持RAD51稳定性以及通过减轻PARPi介导的PARP1滞留来保护同源重组修复位点方面的作用提供了机制上的见解。
