Mechanisms and consequences of inaccurate DNA double-strand break repair

描述（由申请人提供）：DNA双链断裂是细胞生存必须修复的危险病变。最近的研究表明，当精确的修复途径受到损害时，容易出错的断裂修复过程（如替代端连接）就会发生。这些不准确的过程导致与遗传疾病和人类癌症，特别是淋巴瘤和白血病有关的基因组缺失和染色体易位。我们的研究目标是表征不准确的双链断裂修复的分子机制，并确定它们的利用如何导致疾病。为了实现这一点，我们建议阐明翻译DNA聚合酶如何在模型后生动物黑腹果蝇中促进不准确的修复。具体而言，我们将研究DNA聚合酶theta在替代末端连接中的新发现作用，并验证聚合酶theta使用协调的解旋酶和聚合酶活性来促进微同源序列退火的假设。在聚合酶theta中具有区域特异性突变的转基因果蝇将被测试其支持替代末端连接修复的能力。此外，该蛋白的突变型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试验中进行测试。在后续实验中，我们将使用体内报告系统来定义翻译聚合酶eta、zeta和Rev1在同源重组修复DNA双链断裂和间隙中的作用，这些DNA双链断裂和间隙是由I-SceI内切酶表达和转座子切除引起的。我们还将比较需要不同数量DNA合成的复制聚合酶在断裂修复中的作用，并确定它们的损伤如何影响翻译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如何影响替代末端连接，我们将构建嵌入不同DNA重复背景的I-SceI位点的质粒，并在表达I-SceI的胚胎中检测修复。最后，我们将描述在缺乏聚合酶介导的替代末端连接的情况下易于缺失的修复途径，并测试该备份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翻译聚合酶和相关蛋白如何进行断裂DNA的不准确重新连接，并将为容易出错的双链断裂修复如何导致在癌症和相关疾病中经常观察到的基因组不稳定提供重要见解。

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.