Genome Maintenance via the BRCA-RAD54 Axis

通过 BRCA-RAD54 轴进行基因组维护

• 批准号: 10577842
• 负责人: WEIXING Wilson ZHAO
• 金额: $ 32.55万
• 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/10577842
• 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; Health; Human; Impairment; Infertility; Invaded; Joints; Knowledge; Laboratories; Lesion; Licensing; Maintenance; Malignant Neoplasms; Mammals; Mammary Neoplasms; Mediating; Mediator; 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; brca gene; cofactor; ds-DNA; mutant; nervous system disorder; novel; ovarian neoplasm; preservation; presynaptic; protein complex; reconstitution; single molecule; structural biology; synergism; 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 同源指导的DNA修复（HDR）是一种通用的保守机制，用于修复DNA双重 链断裂和DNA复制叉的维护。而HDR相对理解 细菌和较低的真核生物，明显的知识差距仍然在对机制的欣赏中 哺乳动物，由于大大增加了复杂性，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剂，包括BRCA1-BARD1，BRCA2和PALB2 在哺乳动物中的HDR中，新兴证据提供了他们履行的角色的瞥见。但是，整体 了解人HDR的分子机制已受到巨大挑战的阻碍 表达和纯化这些非常大的蛋白质和蛋白质复合物进行机械工作。我的 实验室克服了这一挑战，使我们能够证明BRCA2在复杂的复杂角色中的关键作用 其强制性伴侣DSS1在促进RAD51的催化活性丝组装方面 单链DNA上的重组酶，并通过HDR揭示了BRCA1-BARD1复合物的晚期作用 促进RAD51介导的DNA链入侵。在此提案中，我们旨在填补一些知识空白 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。具体而言，我们将采用一种综合方法，该方法涵盖重建生物化学， 单分子生物物理学，结构生物学，DNA纤维分析和晚期细胞生物学询问 上述肿瘤抑制剂及其伴侣HDR因素（例如RAD54）如何有助于促进 RAD51-SSDNA核蛋白丝的组装和DNA链入侵的催化 染色质的上下文。我们工作的结果将使分子和机械性清晰度达到RAD51-- 依赖的HDR和人类细胞中应力的DNA复制叉的保存。