Mechanisms and consequences of inaccurate DNA double-strand break repair

DNA双链断裂修复不准确的机制和后果

基本信息

批准号：
8107221
负责人：
Mitch McVey
金额：
$ 29.46万
依托单位：
TUFTS UNIVERSITY MEDFORD
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-09-01 至 2016-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8107221
关键词：
Adult Affect Biochemical Biological Assay Cell Survival Cells Chromosomal translocation Chromosomes Collaborations Companions DNA DNA Double Strand Break DNA Repair DNA Sequence DNA Sequence Rearrangement DNA biosynthesis DNA-Directed DNA Polymerase Data Development Disease Double Strand Break Repair Drosophila genus Drosophila melanogaster Embryo Event Excision Gene Mutation Genome Stability Genomic Instability Genomics Goals Hereditary Disease Human Impairment Insecta Investigation Knowledge Lead Lesion Malignant Neoplasms Measures Mediating Modeling Molecular Molecular Genetics Monitor Mutation Nonhomologous DNA End Joining Organism Other Genetics Outcome Pathway interactions Plasmids Point Mutation Polymerase Positioning Attribute Predisposition Process Proteins Recruitment Activity Reporter Research Residual state Role Site Syndrome System Testing Therapeutic Agents Tissues Transgenic Organisms Up-Regulation Variant base cancer cell cancer genetics endodeoxyribonuclease SceI experience fly helicase homologous recombination in vivo insertion/deletion mutation insight leukemia/lymphoma mutant novel prevent protein function recombinational repair reconstitution repaired research study stem cell population tumor tumor initiation tumor progression tumorigenesis

项目摘要

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.

描述（由申请人提供）：DNA双链断裂是危险的病变，必须修复才能生存。最近的研究表明，当准确的修复途径受到损害时，容易发生的断裂修复过程（例如替代端连接）会运行。这些不准确的过程导致基因组缺失和染色体易位，这些易位与遗传疾病和人类癌症有关，尤其是淋巴瘤和白血病。我们研究的目的是表征不准确的双链破裂修复的分子机制，并确定其利用如何导致疾病。为此，我们建议阐明跨果果果蝇果蝇模型中的跨性质DNA聚合酶如何导致不准确的修复。具体而言，我们将研究DNA聚合酶theta在替代末端的新作用，并测试聚合酶theta使用协调的解旋酶和聚合酶活性的假设，以促进微型同学序列退火。在聚合酶theta中具有域特异性突变的转基因果蝇将通过支持替代性最终连接修复的能力进行测试。此外，该蛋白质的突变体和野生型变体将在昆虫细胞中表达，在解旋酶和聚合酶测定中进行纯化和测试。在伴侣实验中，我们将使用体内报告基因系统来定义translesion聚合酶ETA，Zeta和Rev1在DNA双链断裂的同源重组修复中的作用，这些修复是由I-SCEI内核酸酶表达和转孔子切除术引起的。我们还将比较复制性聚合酶在需要各种DNA合成的断裂修复中的作用，并确定它们的损害如何影响在类似的修复环境中使用跨性别聚合酶的利用。为了深入了解局部序列上下文可以影响替代端的连接的方式，我们将使用嵌入在各种DNA重复上下文中的I-SCEI位点构建质粒，并在表达I-SCEI的胚胎中进行测定修复。最后，我们将表征一个容易缺失的修复途径，该途径在没有聚合酶theta介导的替代端连接的情况下运行，并测试该备用途径是主动分裂成人蝇组织的肿瘤发生的主要原因。这些实验将共同定义跨性别聚合酶和相关蛋白如何进行破裂的DNA的重新加入，并将提供重要的洞察力，以了解容易出错的双链破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。公共卫生相关性：癌症和其他遗传疾病可能是由于DNA双螺旋的两条链破裂然后不正确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。该提案的目的是确定涉及不准确修复的蛋白质，并表征其作用如何导致导致肿瘤形成的细胞变化。