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

描述（由申请人提供）：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畸变，从而导致遗传疾病和癌症。研究人员的长期目标是开发方法，以尽量减少DSB导致的基因组不稳定性。为此，必须确定DSB修复是如何执行和调节的，以及它如何导致基因组不稳定。该项目的目的是解开一些能够放大模式生物酿酒酵母中DSB后果的分子机制。首先，该提议集中在染色单体融合上，其通过将其引导到断裂-融合-桥（BFB）循环中来放大单个DSB的不稳定作用，所述断裂-融合-桥循环产生一系列易于再增殖的次级DSB。初步数据使研究者提出，染色单体融合可以通过允许反向DNA重复序列（IR）之间的分子间单链退火（SSA）来刺激DSB。遗传学方法和物理分析的分子中间体，提出了调查这一点，以及其他同源驱动的染色单体融合的途径，目前知之甚少。第二，这项提议将揭示允许断裂染色体获得端粒的机制。初步数据表明，断裂诱导复制（BIR）是发生BFB的染色体稳定的主要机制，这使得BIR成为BFB相关GCR（如缺失、扩增和易位）的主要来源。这项研究将专门研究易位的形成，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的中断或BIR中间体的其他异常加工导致新的染色体断裂，其导致DNA不稳定性的级联，类似于已知的非相互易位（NRT）途径，其放大了哺乳动物中由初始DSB引起的重排的数量。因此，该提议代表了第一个能够模拟哺乳动物NRT的酵母模型，并旨在阐明这一过程的分子机制。此外，将研究遗传和环境因素对引导BIR修复进入GCR产生途径的影响。总之，这项研究将阐明DSB修复可能导致比初始断裂更具破坏性的基因组后果的分子机制。有人提出，染色单体融合，BIR和NRT是三个这样的过程能够放大的风险所造成的DSB主要是由于触发BFB周期。此外，提出实验来测试是否放大的损害，从这些基因组不稳定的DSB修复过程中产生的细胞暴露于各种环境因素可以进一步放大。为此，实验计划测试各种DNA损伤剂的影响，包括抗癌药物，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。 公共卫生相关性：本研究的目的是揭示通过引导双链DNA断裂进入染色体重排导致基因组不稳定的分子机制。由于遗传畸变是癌细胞的标志，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.