Mechanisms of RNA-dependent DNA repair in humans

人类 RNA 依赖性 DNA 修复机制

• 批准号: 10347369
• 负责人: ALEXANDER V MAZIN
• 金额: $ 45.64万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-16 至 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10347369
• 关键词: Affinity; BRCA deficient; Binding; Binding Proteins; Binding Sites; Biochemical; Cells; Complex; Coupled; DNA; DNA Double Strand Break; DNA Repair; DNA Repair Gene; DNA biosynthesis; DNA lesion; DNA-Directed DNA Polymerase; Data; Development; Double Strand Break Repair; Excision; Filament; Genetic; Genetic Transcription; Genome; Goals; Hereditary Breast Carcinoma; Human; Human Genome; Hybrids; In Vitro; Maintenance; Malignant neoplasm of ovary; Mass Spectrum Analysis; Modification; Mutation; Pathway interactions; Play; Post-Translational Protein Processing; Property; Proteins; RAD52 gene; RNA; RNA Binding; RNA annealing; RNA primers; Reaction; Regulation; Role; Saccharomyces cerevisiae; Site; Structure; Yeasts; brca gene; cancer cell; cancer therapy; crosslink; ds-DNA; homologous recombination; improved; in vivo; mutant; neoplastic cell; novel; nucleic acid structure; reconstitution; recruit; repaired; replication factor A; replication stress; response; single molecule; transcriptome sequencing; tumor; yeast two hybrid system

Summary of the project DNA double-strand breaks (DSB), the most harmful type of DNA lesions, are faithfully repaired by Homologous recombination (HR). It is universally accepted that HR uses homologous dsDNA as a template for DSB repair. However, recent studies indicate that homologous RNA can also be utilized by HR. RNA may serve as a template for DSB repair or as a primer in the R-loop structure (three-stranded nucleic acid structure consisting of a DNA-RNA hybrid and the displaced ssDNA strand) during restart of DNA replication stalled at DNA lesions. Since ~75% of human genome are capable of being transcribed, RNA may play a significant role in DNA repair. However, very little is known about the mechanisms of RNA-dependent DSB repair by HR. Recently, we and others found that RAD52 protein plays an important role in RNA-dependent DSB repair in yeast and humans. We showed that RAD52 promotes formation of RNA:DNA hybrids through a novel mechanism: inverse RNA strand exchange. In contrast to the conventional (forward) reaction that is initiated at ssDNA to carry out DNA strand exchange with homologous dsDNA, the inverse reaction is initiated at dsDNA containing DSB to carry out strand exchange with homologous RNA (or ssDNA). RAD52-promoted inverse RNA strand exchange is stimulated by Replication Protein A (RPA), a ubiquitous ssDNA binding protein. In addition, our current data indicate that RPA may have a novel direct role in RNA- dependent DSB repair. We found that RPA binds RNA with high affinity in vitro and forms RPA-RNA complexes in human cells. Furthermore, our data show that RPA can promote formation of R-loops in vitro. being the first known protein that possesses this activity. Using biochemical, cellular, single-molecule, and reconstitution approaches we want to understand the mechanisms of RNA-dependent DDB repair promoted by human RAD52 and RPA and its role in genome maintenance. Our AIMs are to study: 1) the mechanism of RAD52-promoted inverse RNA strand exchange and its role in DNA repair and 2) the role of RPA in RNA-dependent DNA repair. The proposed studies are expected to contribute to our understanding of the mechanisms of DNA repair in humans and will help to identify critical functions of RAD52 and RPA in BRCA1/2-deficient tumor cells for development of new cancer therapies.

项目概要 DNA 双链断裂 (DSB) 是最有害的 DNA 损伤类型，可通过 Homologous 忠实修复 重组（HR）。人们普遍认为 HR 使用同源 dsDNA 作为 DSB 修复的模板。 然而，最近的研究表明同源RNA也可以被HR利用。 RNA 可以作为 用于 DSB 修复的模板或作为 R 环结构中的引物（三链核酸结构，组成 DNA-RNA 杂合体和移位的 ssDNA 链）在 DNA 复制重新启动期间停滞在 DNA 处 病变。由于约 75% 的人类基因组能够被转录，RNA 可能在 DNA修复。然而，人们对 HR 修复 RNA 依赖性 DSB 的机制知之甚少。 最近，我们和其他人发现 RAD52 蛋白在 RNA 依赖性 DSB 修复中发挥重要作用 酵母和人类。我们证明 RAD52 通过一种新的方法促进 RNA:DNA 杂交体的形成 机制：反向RNA链交换。与在以下位置引发的传统（正向）反应相反 ssDNA与同源dsDNA进行DNA链交换，逆反应在dsDNA处启动 含有 DSB 与同源 RNA（或 ssDNA）进行链交换。 RAD52 促进的反向 RNA 链交换受到复制蛋白 A (RPA) 的刺激，RPA 是一种普遍存在的 单链DNA结合蛋白。此外，我们目前的数据表明 RPA 可能在 RNA- 依赖 DSB 修复。我们发现RPA在体外以高亲和力结合RNA并形成RPA-RNA 人体细胞中的复合物。此外，我们的数据表明 RPA 可以促进体外 R 环的形成。 是第一个已知的具有这种活性的蛋白质。 使用生化、细胞、单分子和重构方法，我们希望了解 人RAD52和RPA促进RNA依赖性DDB修复的机制及其在基因组中的作用 维护。我们的目标是研究：1）RAD52促进反向RNA链交换的机制 及其在 DNA 修复中的作用以及 2) RPA 在 RNA 依赖性 DNA 修复中的作用。拟议的研究是 预计将有助于我们了解人类 DNA 修复机制，并将有助于 确定 BRCA1/2 缺陷肿瘤细胞中 RAD52 和 RPA 对新癌症发展的关键功能 疗法。