Double strand break repair maelstrom: causes, mechanisms and genome destabilizing consequences

双链断裂修复漩涡：原因、机制和基因组不稳定后果

• 批准号: 10406966
• 负责人: Anna L Malkova
• 金额: $ 37.78万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-06-01 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10406966
• 关键词: Affect; Algorithms; Area; Automobile Driving; Cell Death; Cell Survival; Cells; Chromosomal Rearrangement; Complex; DNA; DNA Double Strand Break; DNA Repair; DNA Repair Pathway; DNA Sequence Rearrangement; DNA biosynthesis; DNA lesion; Dangerousness; Development; Disease; Double Strand Break Repair; Eukaryota; Event; Experimental Designs; Genetic; Genome; Genome Stability; Genomic Instability; Genomics; Goals; HO nuclease; Human; Human Genome; In Vitro; Joints; Kinetics; Knowledge; Lesion; Malignant Neoplasms; Mediating; Modeling; Molecular; Mutation; Neurologic; Organism; Pathway interactions; Pattern; Plant Roots; Positioning Attribute; Process; Proteins; Regulation; Research; Role; Single-Stranded DNA; Site; Structure; Syndrome; System; Therapeutic Intervention; Work; Yeasts; genetic approach; genome database; high risk; human disease; improved; in vivo; interest; programs; repaired; software development

Maintaining genetic stability is of paramount importance for the survival of cells and organisms. Double-strand DNA breaks (DSBs) are the most lethal DNA lesion threatening genomic stability, and cells have evolved a variety of mechanisms for their repair. While some of the repair mechanisms are accurate, others are “risky” and can further destabilize the genome, leading to cancer and other diseases in humans. The molecular events that draw the intermediates of otherwise accurate repair pathways into a “maelstrom” of destabilizing DNA repair mechanisms, where these intermediates are then processed through risky DNA repair pathways, remain unexplored. The goal of our research is to understand how DSB repair is channeled into the deleterious repair pathways, with particular emphasis on three DSB repair phenomena: 1) break-induced replication (BIR), an unusual type of long-tract repair DNA synthesis that promotes bursts of genetic instabilities; 2) microhomology-mediated BIR (MMBIR), a replicative pathway involving multiple template switching events at positions of microhomologies that yields complex genomic rearrangements; and 3) the transformation of long single-strand DNA intermediates of DSB repair into “toxic” joint molecules promoting cell death. As a starting point, we are using our dependable and powerful system in yeast, where a single DSB is initiated by a site- specific HO endonuclease; we have demonstrated that all three of the repair events of interest can be used to repair the lesion in this system. The knowledge obtained using this system – the repair mechanisms, intermediates, participating proteins, and mutation patterns – is used to inform the experimental design of studies that will evaluate these pathways in other yeast and mammalian systems. Conceptually, the long-term goals are the same across projects and involve three primary areas of inquiry. First, using sensitive genetic approaches, proteins and DNA motifs whose presence affect the funneling of the repair intermediates into the “maelstrom” of destabilizing repair mechanisms will be identified. Second, a combination of in vivo and in vitro approaches will be used to model and investigate the cell's decision points to understand the circumstances (structures, kinetics, participating proteins, etc.) that draw intermediates into high-risk and/or toxic repair pathways. Third, the patterns of mutations and chromosomal rearrangements that result from the deleterious repair pathways will be evaluated, and computational approaches will be used to apply these findings to human genome databases. To this end, MMBIRFinder, new software developed from previous research, will be used to detect complex genetic changes that cannot be found by currently available algorithms. Overall, this research program will bring improved clarity regarding the mechanisms of DNA repair intermediate processing, which will uncover factors that influence the regulation of dangerous repair pathways and result in destabilization of the genome in eukaryotes.

保持遗传稳定性对于细胞和组织的存活至关重要。双链 DNA断裂（DSB）是威胁基因组稳定性的最致命的DNA病变，细胞已经进化 修复的各种机制。虽然某些维修机制是准确的，但另一些是“风险的” 并可能进一步破坏基因组，从而导致人类癌症和其他疾病。分子 将原本准确修复途径的中间体吸引到破坏稳定的“漩涡”的事件 DNA修复机制，然后通过风险的DNA修复途径处理这些中间体， 保持出乎意料。我们研究的目的是了解如何将DSB维修引入有害 修复途径，特别强调三个DSB修复现象：1）破裂引起的复制（BIR）， 一种不寻常的长期修复DNA合成，促进了遗传不稳定性的爆发； 2） 微学介导的BIR（MMBIR），复制途径涉及多个模板切换事件 产生复杂基因组重排的微观学位置； 3）长的转变 DSB修复的单链DNA中间体促进细胞死亡的“有毒”关节分子。作为开始 重点，我们在酵母中使用了我们可靠且功能强大的系统，在该酵母中，单个DSB是由站点启动的 特定的HO核酸内切酶；我们已经证明，所有感兴趣的维修事件都可以用作 修复该系统中的病变。使用该系统获得的知识 - 维修机制， 中间体，参与蛋白质和突变模式 - 用于告知实验设计 将在其他酵母和哺乳动物系统中评估这些途径的研究。从概念上讲，长期 在项目之间的目标是相同的，涉及三个主要探究领域。首先，使用敏感遗传 接近，蛋白质和DNA基序，其存在影响修复中间体的漏斗 将确定破坏维修机制的“漩涡”。第二，体内和体外的组合 方法将用于建模和调查细胞的决策点以了解情况 （结构，动力学，参与蛋白等）将中间体吸引到高风险和/或有毒的维修中 途径。第三，由有害的突变和染色体重排的模式 将评估维修途径，并将使用计算方法将这些发现应用于 人类基因组数据库。为此，从以前的研究开发的新软件mmbirfinder将 用于检测当前可用算法无法找到的复杂遗传变化。总体而言，这 研究计划将提高有关DNA修复中间处理机制的清晰度， 这将发现影响危险维修途径调节的因素，并导致 真核生物中基因组的不稳定。