Mechanisms of error prone repair of DNA breaks

DNA 断裂易错修复机制

• 批准号: 9222761
• 负责人: SANG EUN LEE
• 金额: $ 28.81万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9222761
• 关键词: Address; Bar Codes; Biochemical; Biological; Biological Assay; Biological Models; Bypass; Cancer Etiology; Cell Cycle; Cell Cycle Stage; Cells; Cellular biology; Chromatin; Chromosomal Instability; Chromosomal Rearrangement; Chromosomal translocation; Chromosome Territory; Chromosome abnormality; Chromosomes; Coupled; DNA; DNA Damage; DNA Double Strand Break; DNA Repair; DNA Repair Pathway; DNA Sequence Alteration; DNA replication fork; Development; Direct Repeats; Double Strand Break Repair; Etiology; Event; Frequencies; Gene Deletion; Genes; Genetic; Genetic Recombination; Genetic Screening; Genomic Segment; Genomics; Goals; Hereditary Disease; Homing; Human; Human Genetics; Immunologic Receptors; Kinetics; Knowledge; Lead; Length; Light; Maintenance; Malignant Neoplasms; Mediating; Modeling; Molecular; Molecular Biology; Molecular Genetics; Mutation; Nonhomologous DNA End Joining; Normal Cell; Nuclear; Oncogenic; Outcome; Outcome Study; Pathogenicity; Pathway interactions; Pattern; Plasmids; Process; Proliferating; Regulation; Research; Role; Saccharomycetales; Signal Transduction; Stress; System; TEL1 Gene; Techniques; Testing; Time; Translocation Breakpoint; Tumor Suppression; Vertebrates; Yeasts; activation-induced cytidine deaminase; base; carcinogenesis; deletion library; endodeoxyribonuclease SceI; endonuclease; genome integrity; homologous recombination; human disease; interest; next generation sequencing; novel; public health relevance; repaired; response; telomere

DESCRIPTION (provided by applicant): Microhomology-mediated end joining (MMEJ) repairs DNA breaks by annealing 2-20 bp of flanking microhomology (MH), yielding repair products with deletions of MH and inter-MH sequences. MMEJ is thus a highly error prone repair mechanism with a strong propensity to lead to chromosomal translocations and cancer-causing mutations. Accordingly, the breakpoint junctions of many oncogenic chromosomal translocations feature MH, underscoring the importance of this mechanism for the development chromosome instability and carcinogenesis. Emerging evidence also suggests that MMEJ is an evolutionarily conserved mechanism from yeast to human, and it is involved in the repair of DNA double strand breaks, telomere fusion and immune receptor development. However, we do not know when and where MMEJ operates or how it coordinates and competes against other DNA repair processes. We also do not know if the chromatin and nuclear landscape surrounding DNA breaks impinge on the outcomes of MMEJ and chromosomal aberration formation. It is thus imperative to define the basic mechanism of MMEJ and its genetic and biochemical attributes in a model system with the most tractability. Recently, we have developed both chromosome-based and plasmid-based systems that produce MMEJ repair in budding yeast cells at a high frequency. These systems are most amenable for defining the spatial and temporal patterns of MMEJ and its relationship to canonical repair pathways. Employing these assays, we will test if MMEJ occurs at specific times in the cell cycle and is restricted to a unique nuclear compartment. We will also initiate a powerful genetic screen for new MMEJ genes by combining our plasmid-based MMEJ assay with the bar-coded array or a next generation sequencing technique with the nonessential gene deletion library. Using an approach combining genetics, cell biology and genomic techniques, we also plan to address how MMEJ bypasses end tethering and chromosome territories, two barriers against the formation of chromosomal translocation. Together, the outcomes of this proposal will shed light on the fundamental principles of MMEJ and its contribution to chromosomal instability in many human diseases including cancer.

描述（由申请人提供）：微学介导的末端连接（MMEJ）维修DNA断裂，通过退火2-20 bp侧翼微学学（MH），产生具有MH和MH Inter-MH序列的维修产品。因此，MMEJ是一种高度误差的修复机制，具有很强的倾向，可导致染色体易位和引起癌症的突变。因此，许多致癌染色体易位的断点连接具有MH，强调了这种机制对于发育染色体不稳定性和致癌作用的重要性。新兴的证据还表明，MMEJ是从酵母到人类的进化保守机制，它参与了DNA双链断裂，端粒融合和免疫受体发展的修复。但是，我们不知道MMEJ在何时何地运作，或者它如何坐标和与其他DNA修复过程竞争。我们也不知道DNA周围的染色质和核景观是否会影响MMEJ和染色体畸变形成的结果。因此，必须在模型系统中定义MMEJ的基本机制及其遗传和生化属性。最近，我们开发了基于染色体的和质粒的系统，这些系统以高频在萌芽的酵母细胞中生产MMEJ修复。这些系统最适合定义MMEJ的空间和时间模式及其与规范修复途径的关系。使用这些测定法，我们将测试MMEJ是否在细胞周期中的特定时间发生，并仅限于独特的核室。我们还将通过将基于质粒的MMEJ测定与条形码阵列或下一代测序技术与非必需基因删除库相结合，来启动新MMEJ基因的强大遗传筛选。使用遗传学，细胞生物学和基因组技术结合的方法，我们还计划解决MMEJ如何绕过最终束缚和染色体领土，这是针对染色体易位形成的两个障碍。总之，该提案的结果将阐明MMEJ的基本原理及其对包括癌症在内的许多人类疾病的染色体不稳定性的贡献。