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）：微同源介导的末端连接（MMEJ）通过退火2- 20bp的侧翼微同源（MH）修复DNA断裂，产生MH和MH间序列缺失的修复产物。因此，MMEJ是一种非常容易出错的修复机制，具有导致染色体易位和致癌突变的强烈倾向。因此，许多致癌染色体易位的断点连接处都具有MH特征，强调了这一机制在染色体发育不稳定和致癌中的重要性。新的证据还表明，从酵母到人类，MMEJ是一种进化保守的机制，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何时何地起作用，也不知道它如何协调和竞争其他DNA修复过程。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的结果和染色体畸变的形成。因此，将MMEJ的基本机制及其遗传和生化属性定义在一个最具可追溯性的模型系统中是十分必要的。最近，我们开发了基于染色体和基于质粒的系统，在出芽酵母细胞中以高频率产生MMEJ修复。这些系统最适合用于定义MMEJ的时空模式及其与规范修复途径的关系。利用这些试验，我们将测试MMEJ是否发生在细胞周期的特定时间，是否局限于一个独特的核室。我们还将启动一个强大的基因筛选新的MMEJ基因，通过结合我们的质粒为基础的MMEJ检测与条形码阵列或下一代测序技术与非必需基因缺失文库。利用遗传学、细胞生物学和基因组技术相结合的方法，我们还计划解决MMEJ如何绕过染色体易位形成的两个障碍末端系固和染色体区域。总之，这一提议的结果将揭示MMEJ的基本原理及其在包括癌症在内的许多人类疾病中对染色体不稳定性的贡献。