Mechanisms of error prone repair of DNA breaks

DNA 断裂易错修复机制

• 批准号: 8698570
• 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 2018-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8698570
• 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 Rearrangement; Development; Direct Repeats; Double Strand Break Repair; Etiology; Event; Frequencies; Gene Deletion; Gene Mutation; Genes; Genetic; Genetic Recombination; Genetic Screening; 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; Pathway interactions; Pattern; Plasmids; Process; Proliferating; Regulation; Research; Role; Saccharomycetales; Signal Transduction; Staging; Stress; System; Techniques; Testing; Time; Translocation Breakpoint; Tumor Suppression; Vertebrates; Yeasts; activation-induced cytidine deaminase; base; carcinogenesis; deletion library; endodeoxyribonuclease SceI; endonuclease; 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）：微同源介导的末端连接（MMEJ）通过使侧翼微同源（MH）的2-20bp退火来修复DNA断裂，产生具有MH和MH间序列删除的修复产物。因此，MMEJ 是一种极易出错的修复机制，极有可能导致染色体易位和致癌突变。因此，许多致癌染色体易位的断点连接具有 MH 特征，强调了这种机制对于染色体不稳定和癌发生的重要性。新出现的证据还表明，MMEJ 是从酵母到人类的进化上保守的机制，它参与 DNA 双链断裂的修复、端粒融合和免疫受体的发育。然而，我们不知道 MMEJ 何时何地运作，也不知道它如何与其他 DNA 修复过程协调和竞争。我们也不知道 DNA 断裂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，有必要在最容易处理的模型系统中定义 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和基于质粒的系统，可以在芽殖酵母细胞中高频率地产生 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。利用这些测定，我们将测试 MMEJ 是否发生在细胞周期的特定时间并且仅限于独特的核区室。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文库相结合，启动对新 MMEJ 基因的强大遗传筛选。我们还计划使用结合遗传学、细胞生物学和基因组技术的方法来解决 MMEJ 如何绕过末端束缚和染色体区域（阻止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中染色体不稳定性的贡献。