The Effect of Subnuclear Compartmentalization on DNA Double-Strand Break Repair

亚核区室化对 DNA 双链断裂修复的影响

• 批准号: 10463980
• 负责人: Alyssa Laffitte
• 金额: $ 4.68万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-01 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10463980
• 关键词: Affect; Agreement; Antibodies; Biogenesis; Biological Assay; Biological Models; Cell Cycle; Cell Nucleus; Cells; Centromere; Chromosomes; Complementary RNA; DNA; DNA Double Strand Break; DNA Repair; DNA Repair Pathway; Data; Detection; Disease; Double Strand Break Repair; Educational process of instructing; Enzymes; Event; Excision; Failure; Fission Yeast; G2 Phase; Genetic; Genetic Engineering; Genetic Models; Genetic Predisposition to Disease; Genetic Recombination; Genetic Transcription; Genome Stability; Genomic Segment; Goals; Heterochromatin; Hybrids; Image; Immunoprecipitation; Invaded; Kinetics; Lead; Ligation; Light; Malignant Neoplasms; Measures; Mentors; Methods; Mutation; Nonhomologous DNA End Joining; Nuclear; Outcome; Pathway interactions; Play; Positioning Attribute; Process; Production; Proteins; RAD52 gene; RNA; RNA Degradation; Resected; Resolution; Ribonuclease H; Ribonucleases; Ribosomal DNA; Role; SS DNA BP; Single-Stranded DNA; Sister Chromatid; Site; Small RNA; Source; Testing; Time; Training; Transcript; Work; Writing; Yeast Model System; Yeasts; antagonist; career; design; experience; experimental study; genome integrity; homologous recombination; improved; insight; live cell imaging; overexpression; protein complex; recombinational repair; recruit; repaired; response; skills; transcriptome sequencing

Project Summary/Abstract DNA double-stranded breaks (DSBs) represent a danger to genome stability. Cells can repair DSBs through non-homologous end-joining (NHEJ) or homologous recombination (HR). Repair pathway choice is critical because failure to choose the most faithful repair mechanism can lead to cancer. NHEJ is the direct ligation of the DSB ends, and as such it does not involve any repair templates. It is rapid, but error prone. In contrast, HR is the more faithful repair pathway that uses the sister chromatid as a template for repair and therefore is upregulated in S/G2 phases of the cell cycle. HR is initiated by the end resection of the DSB (creating 3’ single- stranded overhangs). It then proceeds with the loading of repair proteins, most importantly RAD51, onto the resulting ssDNA to allow the broken chromosome to strand invade a homologous template (ideally the sister chromatid). New synthesis and resolution complete homology-dependent repair. My project focuses on how HR is regulated post-resection, especially on how the recruitment of recombination factors is regulated in time and space. Previous work has shown that nuclear compartmentalization plays a role in regulating the loading of repair factors onto resected ssDNA through a mechanism involving RNA-DNA hybrids. Loading of repair factors is different in the two nuclear compartments: the euchromatic nuclear interior and the heterochromatic nuclear periphery. Specifically, in the nuclear interior, Rad52, which remodels RPA to Rad51 loading in yeast, is efficiently recruited to resected DSBs and is retained there during repair. By contrast, in DSBs tethered to the nuclear periphery, Rad52 loading is highly transient. My preliminary data suggest that Rad52 loading is antagonized at the nuclear periphery by hybridization of RNA with the resected ssDNA. Accordingly, reducing RNA/DNA hybrids by over-expression of RNase H partially restores Rad52 loading in DSBs at the nuclear periphery. This suggests a competitive mechanism between RNA-DNA hybrids and loading of pro-HR factors onto resected ssDNA at a DSB specifically at the nuclear periphery. My goal is to identify the source of the relevant RNAs, how they are processed, and how they affect DSB repair. My approach is to use a genetic model to investigate the loading of small RNAs onto resected ssDNA at asite-specific, inducible double- stranded break (DSB). I plan to use live-cell imaging, quantitative PCR, genetic perturbations, small RNA sequencing, and detection of RNA-DNA hybrids by DNA immunoprecipitation (DRIP using the S9.6 antibody). Further, I will use a previously designed imaging assay to observe the timing of Rad52 loading and resection progression in single cells and genetic repair outcome assays. For each of these approaches, I will use fission yeast strains that are genetically engineered to tether the DSB to the nuclear periphery and to overexpress RNase H2 (to degrade RNA-DNA hybrids). These results will shed light on the relationship between nuclear compartments and DNA repair mechanisms and efficiency, providing new insight into genome integrity.

项目摘要/摘要 DNA双链断裂(DSB)对基因组的稳定性是一种威胁。细胞可以通过修复双链断裂 非同源末端连接(NHEJ)或同源重组(HR)。修复途径的选择至关重要 因为没有选择最可靠的修复机制可能会导致癌症。NHEJ是直接结扎的 DSB结束，因此它不涉及任何修复模板。它速度很快，但很容易出错。相比之下，人力资源 是更忠实的修复途径，它使用姐妹染色单体作为修复的模板，因此 在细胞周期的S/G2期上调。HR由DSB的末端切除启动(创建3‘单- 搁浅的悬挑)。然后，它继续将修复蛋白，最重要的是RAD51，加载到 产生的单链DNA允许断裂的染色体链侵入同源模板(理想情况下是姐妹 染色单体)。新的合成和拆分完全同源依赖修复。我的项目重点是如何 HR在术后受到调控，尤其是重组因子的招募如何被及时调控 和空间。以前的工作表明，核区划在调节负荷方面起到了作用 通过一种涉及RNA-DNA杂交体的机制，修复因子作用于切除的单链DNA。装车修理 两个核室的因素不同：等色核内部和异色核 核外围国家。具体地说，在细胞核内部，Rad52将RPA重塑为酵母中的RAD51， 被有效地招募到切除的DSB中，并在修复期间保留在那里。相比之下，在DSB中， 在核外围，Rad52负载是高度瞬变的。我的初步数据显示，Rad52的装载量 通过将RNA与切除的单链DNA杂交，在核周进行拮抗。相应地，减少 过表达核糖核酸酶H的RNA/DNA杂交物部分恢复双链断裂核内Rad52的载量 外围设备。这表明RNA-DNA杂交体和前HR因子的负载之间存在竞争机制 在特定的核边缘的DSB上切除的单链DNA。我的目标是找出 相关的RNA，它们是如何处理的，以及它们如何影响DSB修复。我的方法是用一种基因 研究ASITE特异的、可诱导的双链DNA上的小RNA负载的模型 搁浅中断(DSB)。我计划使用活细胞成像，定量聚合酶链式反应，遗传干扰，小RNA 通过DNA免疫沉淀法(使用S9.6抗体滴定)对RNA-DNA杂交物进行测序和检测。 此外，我将使用先前设计的成像分析来观察RAD52加载和切除的时间 单细胞进展和遗传修复结果分析。对于这些方法中的每一种，我将使用裂变 通过基因工程将DSB连接到核外围并过度表达的酵母菌株 核糖核酸酶H2(降解RNA-DNA杂交物)。这些结果将有助于阐明核与核之间的关系。 间隔和DNA修复机制和效率，提供了对基因组完整性的新见解。