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

项目总结/摘要 DNA双链断裂（DSB）对基因组稳定性构成威胁。细胞可以修复DSB， 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会导致癌症。NHEJ是直接连接 DSB结束，因此它不涉及任何修复模板。它是快速的，但容易出错。相反，HR 是更可靠的修复途径，使用姐妹染色单体作为修复模板，因此， 在细胞周期的S/G2期上调。HR由DSB的末端切除（产生3'单- 搁浅的突出端）。然后，它继续将修复蛋白，最重要的是RAD 51，装载到 产生的ssDNA允许断裂的染色体进入同源模板（理想情况下是姐妹染色体）， 染色单体）。新的合成和解析完全同源依赖修复。我的项目重点是如何 HR在切除后受到调节，尤其是重组因子的募集如何及时调节 和空间.以前的工作表明，核区室化在调节装载中起作用 修复因子通过涉及RNA-DNA杂交的机制作用于切除的ssDNA上。修复加载 因子在两个核室中是不同的：常染色质核内部和异染色质 核外围具体地说，在核内部，Rad 52，它重塑RPA到Rad 51加载在酵母， 被有效地补充到切除的DSB中，并在修复过程中保留在那里。相比之下，在与 在核外围，Rad 52负载是高度瞬时的。我的初步数据表明，拉德52加载是 通过RNA与切除的ssDNA的杂交在核周边拮抗。因此，减少 RNA酶H过表达的RNA/DNA杂合体部分恢复了DSB中Rad 52在核内的负载。 外围这表明RNA-DNA杂合体与促HR因子负载之间存在竞争机制 在DSB处特异性地在核周边处切割到ssDNA上。我的目标是找出 相关的RNA，它们是如何加工的，以及它们如何影响DSB修复。我的方法是用一种基因 模型，以研究小RNA在位点特异性，诱导性双链DNA上的加载。 链断裂（DSB）。我计划用活细胞成像定量PCR基因干扰小RNA 测序，并通过DNA免疫沉淀（使用S9.6抗体的DRIP）检测RNA-DNA杂交体。 此外，我将使用先前设计的成像分析来观察Rad 52加载和切除的时间 在单细胞和遗传修复结果测定中的进展。对于这些方法中的每一种，我都将使用裂变 酵母菌株，其经遗传工程改造以将DSB拴系到核外围并过表达 RNase H2（降解RNA-DNA杂合体）。这些结果将揭示核与核之间的关系。 区室和DNA修复机制和效率，提供了新的见解基因组的完整性。

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.