Genome Maintenance via the BRCA-RAD54 Axis

摘要 同源性指导的DNA修复（HDR）是一种普遍、保守的DNA双链修复机制。 链断裂和DNA复制叉的维持。虽然人类发展报告在 细菌和低等真核生物，在认识生物的机制方面仍然存在明显的知识差距， 哺乳动物，因为大大增加了复杂性，包括无数独特的因素。值得注意的是， 乳腺和卵巢肿瘤抑制因子，包括BRCA 1-BARD 1、BRCA 2和PALB 2， 在哺乳动物的HDR中，新出现的证据提供了它们所扮演的角色的一瞥。然而，一个整体 理解人类HDR的分子机制一直受到以下巨大挑战的阻碍： 表达和纯化这些非常大的蛋白质和蛋白质复合物用于机械后处理。我 实验室已经克服了这一挑战，使我们能够证明BRCA 2在复杂的 它的强制性伙伴DSS 1在促进组装的催化活性长丝的RAD 51 重组酶的单链DNA，并揭示了在HDR中的BRCA 1-BARD 1复合物的晚期作用，通过 促进RAD 51介导的DNA链侵入。在这份提案中，我们旨在填补几个知识空白 关于BRCA 1-BARD 1、BRCA 2-DSS 1和PALB 2如何在HDR的两个不同阶段协同作用， 过程具体来说，我们将采用一种综合方法，包括重建生物化学， 单分子生物物理学、结构生物学、DNA纤维分析和高级细胞生物学， 上述肿瘤抑制因子及其伴侣HDR因子，如RAD 54，如何帮助促进 RAD 51-ssDNA核蛋白丝的组装和DNA链侵入细胞内的催化作用 染色质的背景。我们工作的结果将为RAD 51带来分子和机制上的清晰度。 依赖性HDR和人类细胞中应激DNA复制叉的保存。

ABSTRACT Homology-directed DNA repair (HDR) is a universal, conserved mechanism for the repair of DNA double- strand breaks and the maintenance of DNA replication forks. While HDR is relatively well understood in bacteria and lower eukaryotes, glaring knowledge gaps remain in the appreciation of mechanisms in mammals, because of greatly added complexity including myriad factors that are unique. Notably, several breast and ovarian tumor suppressors, including BRCA1-BARD1, BRCA2, and PALB2, have been implicated in HDR in mammals, and emerging evidence provides glimpses of the roles that they fulfill. However, a holistic understanding the molecular mechanisms of human HDR has been hampered by the immense challenge of expressing and purifying these very large proteins and protein complexes for mechanistic workup. My laboratory has overcome this challenge, allowing us to demonstrate the pivotal role of BRCA2 in complex with its obligatory partner DSS1 in facilitating the assembly of catalytically active filaments of the RAD51 recombinase on single-stranded DNA and to reveal a late role of the BRCA1-BARD1 complex in HDR via the promotion of RAD51-mediated DNA strand invasion. In this proposal, we aim to fill several knowledge gaps regarding how BRCA1-BARD1, BRCA2-DSS1, and PALB2 synergize in two distinct stages of the HDR process. Specifically, we will apply an integrated approach that encompasses reconstitution biochemistry, single-molecule biophysics, structural biology, DNA fiber analysis, and advanced cell biology to interrogate how the aforementioned tumor suppressors and their partner HDR factors, such as RAD54, help promote the assembly of the RAD51-ssDNA nucleoprotein filament and the catalysis of DNA strand invasion within the context of chromatin. The results from our work will bring molecular and mechanistic clarity to RAD51- dependent HDR and the preservation of stressed DNA replication forks in human cells.