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