Molecular Mechanisms Underlying Recombination at DNA Double-Strand Breaks and Stalled Replication Forks

DNA 双链断裂和停滞复制叉重组的分子机制

• 批准号: 10582329
• 负责人: Rodney J. ROTHSTEIN
• 金额: $ 19.97万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2026-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10582329
• 关键词: Appearance; Biological; Biological Models; Biological Process; Cells; Cellular biology; Chromosomes; Clustered Regularly Interspaced Short Palindromic Repeats; DNA; DNA Damage; DNA Double Strand Break; DNA Repair; DNA biosynthesis; DNA replication fork; DNA-Directed DNA Polymerase; Diploid Cells; Disease; Disease model; Double Strand Break Repair; Excision; Gamma Rays; Genes; Genetic; Genetic Recombination; Genome; Genomic Instability; Homologous Gene; Human; Immunoglobulin Switch Recombination; Lesion; Malignant Neoplasms; Measures; Messenger RNA; Methods; Molecular; Molecular Biology; Movement; Mutation; Organism; Process; Proteins; Role; SMARCA3 gene; Site; Source; System; Techniques; Time; Visualization; Yeasts; assault; cancer cell; design; endonuclease; gene function; gene product; gene replacement; human disease; in vivo; insight; mutant; overexpression; oxidation; repaired

Project Summary Genetic recombination is an essential biological process that is central to the repair of DNA damage that occurs during replication and other assaults on the genome. Unrepaired double-strand breaks (DSBs) are one of the most lethal kinds of DNA damage and their aberrant repair often leads to genome instability, one of the hallmarks of cancer. The damage can occur from endogenous sources, such as oxidation, or from external sources, such as gamma rays. Genetic recombination is also the mechanism for gene replacement and the repair of CRISPR-Cas-induced DSBs, processes that are commonly used to study all diseases in humans as well as human disease models in simpler systems. In this proposal, the mechanisms by which DSBs are repaired will be explored in vivo in yeast cells using a combination of cell biology, genetics and molecular biology. A system to study the repair of the two ends of a DSB has been designed to allow, for the first time, a view into the fate of these ends during the recombination process in diploid cells. This system will be used to assess chromosome end movement during the repair process in both wild type and mutant genetic backgrounds. The DSBs will be introduced at the same site using three different endonucleases that create 3’ overhangs, 5’ overhangs or blunt ends to determine how the repair system processes these different lesions. These same ends will be examined for repair using molecular biological techniques designed to measure (1) end resection, an important step in preparing the DSB end for recombination and (2) the repair synthesis stimulated at the end, which is a necessary step on the path to repair of the break. Another system will be designed to permit in vivo visualization of the first appearance of a successfully recombined chromosome using messenger RNA as surrogate. This system will be combined with various mutations in recombination genes to define when the timing of those gene products are important for the repair of a site-specific DSB. Broken DNA ends also arise during DNA replication, especially when the DNA polymerase is confronted with a nick. A system will be designed to induce a site-specific nick and will be used in conjunction with marked chromosome ends to define the fate of a DSB that is generated after replication of a nick. Finally, the function of the Rad5 protein, a homolog of the human HLTF that is often overexpressed in cancer cells, will be studied to determine its role in template switch recombination. It is known that both HLTF and Rad5 are necessary for recombination during template switch repair, but it is not known whether they are sufficient. By approaching the study of recombination in a genetically tractable system that is easy to manipulate, insights into the conserved mechanisms of recombination will be achieved.

项目摘要 遗传重组是必不可少的生物学过程，是修复DNA损伤的核心 发生在复制期间和对基因组的其他攻击。未修复的双链断裂（DSB）是一个 最致命的DNA损伤及其异常修复通常会导致基因组不稳定性，其中之一 癌症的标志。内源性来源（例如氧化或外部）可能造成损害 来源，例如伽马射线。遗传重组也是基因替代的机制和 修复CRISPR-CAS诱导的DSB，这些过程通常用于研究人类的所有疾病 以及更简单系统中的人类疾病模型。在此提案中，DSB的机制 修复的将在酵母细胞中使用细胞生物学，遗传学和分子的结合在体内探索 生物学。研究DSB两端修复的系统旨在首次允许 在二倍体细胞的重组过程中查看这些末端的命运。该系统将用于 在维修过程中评估野生型和突变通用的染色体末端运动 背景。 DSB将在同一站点使用三种不同的内切酶引入3' 悬垂，5英尺或钝的结束，以确定修复系统如何处理这些不同的病变。 使用旨在测量的分子生物学技术，将检查这些相同的末端以进行修复（1） 末端切除术，准备重组DSB端的重要步骤，以及（2）修复合成 在末尾刺激，这是修复断裂路径的必要步骤。另一个系统将是 旨在允许体内可视化成功重组染色体的首次出现 使用Messenger RNA作为代理。该系统将与重组中的各种突变结合 这些基因产物的时机何时对位点特异性DSB的修复很重要。 在DNA复制过程中，DNA末端也破裂，尤其是当DNA聚合酶面对A 缺口。将设计一个系统来诱导特定地点的刻痕，并将与标记结合使用 染色体结束，以定义复制划痕后生成的DSB的命运。最后，功能 RAD5蛋白是癌细胞中通常过表达的人类HLTF的同源物的。 确定其在模板开关重组中的作用。众所周知，HLTF和Rad5都是必需的 模板开关修复期间的重组，但尚不清楚它们是否足够。通过接近 在易于操纵的一般可拖动系统中的重组研究，对保守的见解 将实现重组的机制。