Amplification of Risk Caused by Mis-Routing of DNA Double-Strand Break Repair

描述(申请人提供)：双链DNA断裂(DSB)对人类健康是危险的，因为不精确或错误的修复通常会导致突变和染色体异常，导致遗传病和癌症。研究人员的长期目标是开发出将DSB引起的基因组不稳定性降至最低的方法。为了达到这个目的，必须确定DSB修复是如何执行和调节的，以及它是如何导致基因组不稳定的。该项目的目的是揭示一些能够在模式生物酿酒酵母中放大DSB后果的分子机制。首先，这个建议集中在染色单体融合上，它通过将单个DSB引导到断裂-融合-桥(BFB)循环中，从而放大单个DSB的不稳定效应，从而产生一系列易于重排的次级DSB。初步数据允许研究人员提出，双链断裂可以通过允许反向DNA重复序列(IR)之间的分子间单链退火(SSA)来刺激染色单体融合。遗传方法和分子中间体的物理分析被提出来研究这一点，以及目前知之甚少的其他同源驱动的染色单体融合途径。其次，这项提议将解开允许断裂的染色体获得端粒的机制。初步数据表明，断裂诱导复制(BIR)是BFBS过程中染色体稳定的主要机制，这使得BIR成为BFB相关GCRs的主要来源，如缺失、扩增和易位。这项研究将专门研究移位的形成，这是BIR最有害的结果。最后，来自遗传学研究的结果导致了这样的假设，即BIR或BIR中间体的其他异常加工的中断会导致新的染色体断裂，从而导致DNA不稳定的级联反应，类似于已知的非相互易位(NRT)途径，该途径会放大哺乳动物最初DSB导致的重排数量。因此，这项提议代表了第一个能够模拟哺乳动物NRT的酵母模型，并旨在揭示这一过程的分子机制。此外，还将研究遗传和环境因素对BIR修复进入GCR产生途径的影响。总之，这项研究将阐明DSB修复可能导致比最初破坏更具破坏性的基因组后果的分子机制。有人认为，染色单体融合、BIR和NRT是三个这样的过程，能够放大DSB引起的风险，主要是由于触发BFB周期。此外，还建议进行实验，以测试这些破坏基因组稳定的DSB修复过程所导致的损伤放大程度是否会因细胞暴露于各种环境因素而进一步放大。为此，计划进行实验，以测试包括抗癌药物在内的各种DNA损伤剂的效果，以调查这些药物是否可能增加高风险修复过程的频率或以其他方式改变其结果。与公共卫生相关：这项研究旨在通过将双链DNA断裂引导到染色体重排来揭开导致基因组不稳定的分子机制。由于基因异常是癌细胞的标志，这项研究将进一步加深我们对某些癌症病因的理解。

DESCRIPTION (provided by applicant): Double-strand DNA breaks (DSBs) are dangerous for human health because imprecise or faulty repair often leads to mutations and chromosome aberrations causing genetic diseases and cancer. The long-term goal of the investigator is to develop ways to minimize genomic instability resulting from DSBs. It is essential for this purpose to establish how DSB repair is executed and regulated, and how it leads to genome destabilization. The aim of this project is to unravel a number of molecular mechanisms capable of amplifying the consequences of DSBs in the model organism Saccharomyces cerevisiae. Firstly, this proposal is focused on chromatid fusions, which amplify the destabilizing effect of a single DSB by channeling it into breakage-fusion- bridge (BFB) cycles that create a series of rearrangement-prone secondary DSBs. Preliminary data allowed the investigator to propose that chromatid fusions can be stimulated by DSBs by allowing inter-molecular single-strand annealing (SSA) between inverted DNA repeats (IRs). Genetic methods and physical analyses of molecular intermediates are proposed to investigate this, as well as other homology-driven pathways of chromatid fusions that are currently poorly understood. Second, this proposal will unravel the mechanisms that allow broken chromosomes to acquire telomeres. Preliminary data suggested that break-induced replication (BIR) is the primary mechanism by which chromosomes undergoing BFBs are stabilized, which makes BIR the primary source of BFB-associated GCRs such as deletions, amplifications, and translocations. This research will specifically investigate the formation of translocations, which is the most deleterious outcome of BIR. Finally, results from genetic studies led to the hypothesis that interruption of BIR or other aberrant processing of BIR intermediates results in new chromosomal breakages that lead to cascades of DNA instability similar to the non-reciprocal translocations (NRTs) pathway known to amplify the number of rearrangements that result from an initial DSB in mammals. Thus, this proposal represents the first yeast model capable of simulating mammalian NRTs and is intended to unravel the molecular mechanisms of this process. In addition, the effects of genetic and environmental factors on channeling BIR repair into the GCR-producing pathways will be investigated. In summary, this research will elucidate the molecular mechanisms by which DSB repair can result in genomic consequences more destructive than the initial breakage. It is proposed that chromatid fusions, BIR, and NRTs are three such processes capable of amplifying the risks caused by a DSB due primarily to triggering BFB cycles. Further, experiments are proposed to test whether the magnification of damage that results from these genome-destabilizing DSB repair processes could be further amplified by cellular exposure to various environmental factors. To this end, experiments are planned to test the effects of various DNA damaging agents, including anti-cancer drugs, to investigate whether these agents might increase the frequency of high-risk repair processes or otherwise alter their outcomes. PUBLIC HEALTH RELEVANCE: This research is aimed to unravel the molecular mechanisms that lead to genomic destabilization by channeling double-strand DNA breaks into chromosomal rearrangements. Because genetic aberrations are a hallmark of cancer cells, this research will further our understanding of the etiology of some cancers.