Mechanisms of RNA-dependent DNA repair in humans

人类 RNA 依赖性 DNA 修复机制

• 批准号: 10396741
• 负责人: ALEXANDER V MAZIN
• 金额: $ 45.52万
• 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/10396741
• 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.

项目总结 DNA双链断裂(DSB)是最具危害性的DNA损伤类型，由同源DNA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可以作为一种 用于DSB修复的模板或作为R-环结构(三链核酸结构包括 DNA-RNA杂交体和移位的ssDNA链)在DNA复制停止的重新开始期间 损伤。由于~75%的人类基因组能够被转录，RNA可能在 DNA修复。然而，目前对HR依赖RNA的DSB修复机制知之甚少。 最近，我们和其他人发现RAD52蛋白在RNA依赖的DSB修复中发挥重要作用。 酵母和人类。我们发现RAD52通过一种新的方式促进RNA：DNA杂交体的形成 机制：反向RNA链交换。与传统的(正向)反应相比，在 与同源dsDNA进行dna链交换，反向反应在dsDNA处开始。 含有DSB与同源RNA(或单链DNA)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中具有新的直接作用。 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 人类细胞中的复合体。此外，我们的数据表明，RPA在体外可以促进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、单分子和重建方法，我们想要了解 人RAD52和RPA促进RNA依赖的DDB修复机制及其在基因组中的作用 维修。我们的目的是研究：1)RAD52促进反向RNA链交换的机制 以及它在DNA修复中的作用；2)RPA在依赖RNA的DNA修复中的作用。建议进行的研究包括 有望有助于我们了解人类DNA修复的机制，并将有助于 确定BRCA1/2缺陷肿瘤细胞中RAD52和RPA在新肿瘤发生中的关键功能 治疗。