Genome Maintenance via the BRCA-RAD54 Axis

摘要 同源定向DNA修复(HDR)是一种普遍的、保守的DNA双链修复机制。 链断裂和DNA复制叉的维护。虽然HDR相对较好地理解了 细菌和低等真核生物，在对其机制的认识上仍然存在明显的知识空白 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，有几个 乳腺癌和卵巢肿瘤抑制基因，包括BRCA1-BARD1，BRCA2和PALB2，已经被涉及 在哺乳动物的HDR中，新的证据提供了它们所扮演的角色的一瞥。然而，一个整体的 人类HDR的分子机制的理解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体，用于机械训练。我的 实验室已经克服了这一挑战，使我们能够展示BRCA2在复杂的 它在促进RAD51催化活性细丝组装中的强制性伙伴DSS1 重组酶在单链DNA上的作用并揭示BRCA1-BARD1复合体在HDR中的晚期作用 促进RAD51介导的DNA链侵袭。在这项提案中，我们的目标是填补几个知识空白 关于BRCA1-BARD1、BRCA2-DSS1和PALB2如何在人类发展报告的两个不同阶段进行协同 进程。具体地说，我们将应用一种综合方法，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伙伴HDR因子，如RAD54，如何帮助促进 RAD51-ssDNA核蛋白微丝的组装及其对DNA链侵袭的催化作用 染色质的背景。我们的工作结果将为RAD51带来分子和机械上的清晰度- 依赖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.