Genome Maintenance via the BRCA-RAD54 Axis.

通过 BRCA-RAD54 轴进行基因组维护。

• 批准号: 10584136
• 负责人: WEIXING Wilson ZHAO
• 金额: $ 4.42万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-06-01 至 2026-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10584136
• 关键词: ATP phosphohydrolase; Address; Affect; Affinity; Aging; Attenuated; BARD1 gene; BRCA1 gene; BRCA2 gene; Bacteria; Binding; Biochemical; Biochemistry; Biological Assay; Biophysics; Breast; Catalysis; Cells; Cellular biology; Chemicals; Chromatids; Chromatin; Chromosomes; Complex; DNA; DNA Binding; DNA Double Strand Break; DNA Maintenance; DNA Mimicry; DNA Repair; DNA Repair Disorder; DNA Repair Gene; DNA replication fork; Double Strand Break Repair; Eukaryota; Excision; Fiber; Filament; Genetic Recombination; Genome; Glare; Health; Human; Impairment; Infertility; Joints; Knowledge; Laboratories; Lesion; Licensing; Light; Maintenance; Malignant Neoplasms; Mammals; Mammary Neoplasms; Mediating; Mediator of activation protein; Modeling; Molecular; Nucleoproteins; Nucleosomes; Organ; Ovary; PALB2 gene; Play; Positioning Attribute; Postdoctoral Fellow; Preparation; Process; Proteins; Publishing; Rad51 recombinase; Radiation; Reaction; Regulation; Research Project Grants; Role; SS DNA BP; Saccharomyces cerevisiae; Saccharomycetales; Sequence Homologs; Single-Stranded DNA; Source; Stress; Structure; Synapses; System; Tail; Testing; Tumor Suppressor Proteins; Work; base; brca gene; cofactor; ds-DNA; mutant; nervous system disorder; novel; ovarian neoplasm; parent grant; preservation; presynaptic; protein complex; reconstitution; single molecule; structural biology; tumorigenesis

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.

抽象的 同源定向 DNA 修复 (HDR) 是一种通用的、保守的 DNA 双链修复机制。 链断裂和 DNA 复制叉的维持。虽然 HDR 在以下领域得到了相对较好的理解： 细菌和低等真核生物，在了解细菌和低等真核生物的机制方面仍然存在明显的知识差距 哺乳动物，因为其复杂性大大增加，包括无数独特的因素。值得注意的是，几个 乳腺癌和卵巢肿瘤抑制基因，包括 BRCA1-BARD1、BRCA2 和 PALB2，与此有关 在哺乳动物的 HDR 中，新出现的证据让我们得以一睹它们所扮演的角色。然而，一个整体 对人类 HDR 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物以进行机械后处理。我的 实验室克服了这一挑战，使我们能够证明 BRCA2 在复杂的疾病中的关键作用 其义务合作伙伴 DSS1 促进 RAD51 催化活性丝的组装 单链 DNA 上的重组酶，并通过 促进 RAD51 介导的 DNA 链入侵。在这个提案中，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 HDR 的两个不同阶段协同作用 过程。具体来说，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 纤维分析和先进细胞生物学来探究 上述肿瘤抑制因子及其伙伴 HDR 因子（例如 RAD54）如何帮助促进 RAD51-ssDNA核蛋白丝的组装和DNA链入侵的催化 染色质的上下文。我们的工作结果将为 RAD51 带来分子和机制的清晰度 依赖的 HDR 和人类细胞中受应激的 DNA 复制叉的保存。