Mechanisms and consequences of inaccurate DNA double-strand break repair

描述（由申请人提供）：DNA双链断裂是危险的病变，必须修复细胞才能生存。最近的研究表明，当准确的修复途径受到损害时，易出错的断裂修复过程，如替代末端连接。这些不准确的过程导致基因组缺失和染色体易位，与遗传疾病和人类癌症，特别是淋巴瘤和白血病有关。我们研究的目标是描述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translesion DNA聚合酶如何有助于不准确的修复模型后生动物果蝇。具体来说，我们将调查一个新发现的作用，DNA聚合酶θ在替代结束连接和测试的假设，聚合酶θ使用协调解旋酶和聚合酶的活动，以促进退火微同源序列。将测试在聚合酶theta中具有结构域特异性突变的转基因果蝇支持替代性末端连接修复的能力。此外，蛋白质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测定中进行测试。在配套实验中，我们将使用体内报告系统来定义translesion聚合酶eta，zeta和Rev 1在同源重组修复DNA双链断裂和缺口，导致I-SceI内切酶表达和转座子切除中的作用。我们还将比较复制型聚合酶在需要不同数量的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环境可以影响替代末端连接的方式，我们将构建具有嵌入各种DNA重复序列环境中的I-SceI位点的质粒，并在表达I-SceI的胚胎中测定修复。最后，我们将描述一个删除倾向的修复途径，在没有聚合酶θ介导的替代末端连接和测试模型，这种备份途径是一个主要的原因，肿瘤发生在成年苍蝇积极分裂组织。总之，这些实验将有助于确定translesion聚合酶和相关蛋白质如何进行断裂DNA的不准确重新连接，并将提供重要的见解，以了解易错双链断裂修复如何导致癌症和相关疾病中经常观察到的基因组不稳定性。

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.