Mechanisms of RNA-dependent DNA repair in humans

人类 RNA 依赖性 DNA 修复机制

• 批准号: 10576319
• 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 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10576319
• 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; Invaded; 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; Single-Stranded DNA; Site; Structure; Yeasts; brca gene; cancer cell; cancer therapy; crosslink; ds-DNA; homologous recombination; improved; in vivo; mutant; neoplastic cell; novel; nucleic acid structure; protein purification; 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，可被Homoprotein忠实地修复 重组（HR）。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。 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， 在DNA复制重新启动期间，DNA-RNA杂交体和置换的ssDNA链的DNA链在DNA 病变由于约75%的人类基因组能够被转录，因此RNA可能在人类基因组中起重要作用。 DNA修复然而，很少有人知道的RNA依赖的DSB修复的HR的机制。 最近，我们和其他人发现，RAD 52蛋白在RNA依赖的DSB修复中起着重要作用。 酵母和人类我们发现，RAD 52通过一种新的途径促进RNA：DNA杂交体的形成。 机制：反向RNA链交换。与常规的（正向）反应相比， 为了与同源dsDNA进行DNA链交换，在dsDNA处引发逆反应 含有DSB以与同源RNA（或ssDNA）进行链交换。 RAD 52促进的反向RNA链交换受到复制蛋白A（RPA）的刺激，RPA是一种普遍存在的蛋白质。 ssDNA结合蛋白。此外，我们目前的数据表明，RPA可能在RNA中有一个新的直接作用， 依赖性DSB修复我们发现RPA在体外与RNA有很高的亲和力结合，形成RPA-RNA 人类细胞中的复合物。此外，我们的数据表明RPA可以促进体外R环的形成。 这是第一个已知的具有这种活性的蛋白质。 使用生物化学，细胞，单分子和重建方法，我们希望了解 人RAD 52和RPA促进RNA依赖的DDB修复机制及其在基因组中的作用 上维护我们的目标是研究：1）RAD 52促进RNA反向链交换的机制 及其在DNA修复中的作用; 2）RPA在RNA依赖性DNA修复中的作用。拟议的研究是 预计将有助于我们理解人类DNA修复的机制，并将有助于 鉴定RAD 52和RPA在BRCA 1/2缺陷肿瘤细胞中对于新癌症发展的关键功能 治疗