Mechanisms and consequences of inaccurate DNA double-strand break repair

描述(申请人提供)：DNA双链断裂是一种危险的损伤，必须修复才能使细胞存活。最近的研究表明，当准确的修复途径受到损害时，容易出错的断裂修复过程，如替代末端连接，就会运作。这些不准确的过程导致基因组缺失和染色体易位，与遗传病和人类癌症有关，特别是淋巴瘤和白血病。我们研究的目的是描述不准确的双链断裂修复的分子机制，并确定它们的使用如何导致疾病。为了实现这一点，我们建议阐明跨损伤DNA聚合酶如何有助于后生动物果蝇模型的不准确修复。具体地说，我们将调查新发现的DNA聚合酶theta在交替末端连接中的作用，并测试聚合酶theta使用协调的解旋酶和聚合酶活性来促进微同源序列的退火的假设。具有聚合酶theta结构域特定突变的转基因果蝇将接受支持替代末端连接修复的能力测试。此外，该蛋白的突变体和野生型变体将在昆虫细胞中表达、纯化并在解旋酶和聚合酶分析中进行测试。在配套实验中，我们将使用体内报告系统来确定跨损伤聚合酶ETA、Zeta和Rev1在I-SCEI内切酶表达和转座子切除引起的DNA双链断裂和缺口的同源重组修复中的作用。我们还将比较复制聚合酶在需要不同数量DNA合成的断裂修复中的作用，并确定它们的损伤如何影响跨损伤聚合酶在类似修复环境中的利用。为了深入了解局部序列背景影响选择性末端连接的方式，我们将构建I-SCEI位点嵌入各种DNA重复背景的质粒，并检测表达I-SCEI的胚胎的修复。最后，我们将描述一种在没有聚合酶theta介导的替代末端连接的情况下操作的易于缺失的修复途径，并测试这一备用途径是成年果蝇活跃分裂组织中肿瘤发生的主要原因的模型。总之，这些实验将有助于定义跨损伤聚合酶和相关蛋白质如何对断裂的DNA进行不准确的重新连接，并将为容易出错的双链断裂修复如何导致癌症和相关疾病中常见的基因组不稳定提供重要的见解。 与公共卫生相关：癌症和其他遗传疾病可能是由DNA双螺旋的两条链断裂，然后错误修复时发生的变化引起的。令人惊讶的是，修复DNA断裂的某些过程往往是不准确的。这项提议的目标是识别参与不准确修复的蛋白质，并表征它们的行为如何导致导致肿瘤形成的细胞变化。

DESCRIPTION (provided by applicant): DNA double-strand breaks are dangerous lesions that must be repaired for cells to survive. Recent studies have demonstrated that error-prone break repair processes such as alternative end joining operate when accurate repair pathways are compromised. These inaccurate processes lead to genomic deletions and chromosome translocations that are associated with genetic diseases and human cancers, especially lymphomas and leukemias. The goal of our research is to characterize molecular mechanisms of inaccurate double-strand break repair and to determine how their utilization may lead to disease. To accomplish this, we propose to elucidate how translesion DNA polymerases contribute to inaccurate repair in the model metazoan Drosophila melanogaster. Specifically, we will investigate a newly-discovered role for DNA polymerase theta in alternative end joining and test the hypothesis that polymerase theta uses coordinated helicase and polymerase activities to promote annealing at microhomologous sequences. Transgenic flies with domain-specific mutations in polymerase theta will be tested for their ability to support alternative end-joining repair. In addition, mutant and wild-type variants of the protein will be expressed in insect cells, purified, and tested in helicase and polymerase assays. In companion experiments, we will use in vivo reporter systems to define the roles of translesion polymerases eta, zeta, and Rev1 in homologous recombination repair of DNA double-strand breaks and gaps that result from I-SceI endonuclease expression and transposon excision. We will also compare the roles of replicative polymerases in break repair requiring various amounts of DNA synthesis and determine how their impairment affects the utilization of translesion polymerases in similar repair contexts. To gain insight into the ways in which local sequence context can affect alternative end joining, we will construct plasmids with I-SceI sites embedded in various DNA repeat contexts and assay repair in embryos expressing I- SceI. Finally, we will characterize a deletion-prone repair pathway that operates in the absence of polymerase theta-mediated alternative end joining and test the model that this backup pathway is a major cause of tumorigenesis in actively dividing tissues of adult flies. Together, these experiments will serve to define how translesion polymerases and associated proteins carry out inaccurate rejoining of broken DNA and will provide important insight into how error-prone double-strand break repair can cause genome instability frequently observed in cancer and related diseases. PUBLIC HEALTH RELEVANCE: Cancer and other genetic diseases can be caused by changes that occur when both strands of a DNA double helix are broken and then repaired incorrectly. Surprisingly, certain processes that repair DNA breaks tend to be inaccurate. The goal of this proposal is to identify proteins involved in inaccurate repair and characterize how their action may lead to cellular changes that cause tumor formation.