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.

项目摘要 DNA双链断裂（DSB），最有害的DNA病变类型 重组（HR）。人力资源使用同源dsDNA作为DSB修复模板是普遍接受的。 但是，最近的研究表明，HR也可以利用同源RNA。 RNA可以用作 用于DSB修复的模板或R环结构中的底漆（三链核酸结构组成 在DNA停滞不前的DNA复制重新启动期间，DNA-RNA杂种和移位的ssDNA链 病变。由于约有75％的人类基因组能够被转录，因此RNA可能在 DNA修复。但是，HR对RNA依赖性DSB修复的机制知之甚少。 最近，我们和其他人发现Rad52蛋白在RNA依赖性DSB修复中起重要作用 酵母和人类。我们表明RAD52通过新颖的 机理：逆RNA链交换。与在 ssDNA与同源dsDNA进行DNA链交换，在DSDNA启动了反应 包含DSB以使用同源RNA（或ssDNA）进行链交换。 RAD52促进的逆RNA链交换通过无处不在的复制蛋白A（RPA）刺激 ssDNA结合蛋白。此外，我们当前的数据表明RPA在RNA-中可能具有新颖的直接作用 依赖的DSB修复。我们发现RPA在体外与高亲和力结合RNA，并形成RPA-RNA 人类细胞中的复合物。此外，我们的数据表明，RPA可以在体外促进R环的形成。 是拥有该活性的第一个已知蛋白质。 使用生化，细胞，单分子和重建方法，我们想了解 由人Rad52和RPA促进的RNA依赖性DDB修复机制及其在基因组中的作用 维护。我们的目的是研究：1）RAD52促进的反RNA链交换的机制 及其在DNA修复中的作用和2）RPA在RNA依赖性DNA修复中的作用。拟议的研究是 有望有助于我们对人类DNA修复机制的理解，并将有助于 确定在BRCA1/2缺陷肿瘤细胞中RAD52和RPA的关键功能以开发新癌症 疗法。