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: 10387418
• 负责人: Anna L Malkova
• 金额: $ 9.9万
• 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/10387418
• 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; Funding Opportunities; 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.

保持遗传稳定性对细胞和生物体的生存至关重要。双链 DNA断裂（DSB）是威胁基因组稳定性的最致命的DNA损伤，细胞已经进化出一种 各种修复机制。虽然有些修复机制是准确的，但其他修复机制则是“有风险的” 并进一步破坏基因组的稳定性，导致人类癌症和其他疾病。分子 这些事件将原本精确的修复途径的中间产物拖入了一个破坏稳定的“漩涡”， DNA修复机制，其中这些中间体然后通过危险的DNA修复途径进行处理， 仍然未被探索。我们研究的目标是了解DSB修复是如何引导到有害的 修复途径，特别强调三种DSB修复现象：1）断裂诱导复制（BIR）， 一种不寻常的长道修复DNA合成类型，促进遗传不稳定性的爆发; 2） 微同源介导的BIR（MMBIR），一种涉及多个模板转换事件的复制途径， 微同源性的位置，产生复杂的基因组重排;和3）长 DSB的单链DNA中间体修复成“毒性”关节分子，促进细胞死亡。为出发 点，我们正在使用我们的可靠和强大的系统在酵母，其中一个单一的DSB是由一个网站启动- 我们已经证明，所有三种感兴趣的修复事件都可以用于 修复这个系统的损伤使用该系统获得的知识-修复机制， 中间体，参与蛋白质和突变模式-用于通知实验设计， 这些研究将在其他酵母和哺乳动物系统中评估这些途径。从概念上讲，长期 所有项目的目标都是相同的，并涉及三个主要的调查领域。首先，使用敏感的基因 方法，蛋白质和DNA基序，其存在影响修复中间体进入细胞的功能， 将识别不稳定修复机制的“漩涡”。其次，体内与体外相结合 方法将被用来建模和调查细胞的决策点，以了解情况 （结构、动力学、参与蛋白质等）将中间体引入高风险和/或毒性修复 途径。第三，由有害基因引起的突变和染色体重排的模式， 将评估修复途径，并使用计算方法将这些发现应用于 人类基因组数据库。为此，从以前的研究中开发的新软件MMBIRcycle将 用于检测目前可用算法无法发现的复杂遗传变化。总体而言，这 研究计划将使DNA修复中间过程的机制更加清晰， 这将揭示影响危险修复途径调节的因素， 真核生物基因组的不稳定。