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

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 它们的修复机制多种多样。虽然有些修复机制是准确的，但另一些机制是“危险的” 并可能进一步破坏基因组的稳定，导致人类患癌症和其他疾病。分子 将原本准确的修复途径的中间环节拖入破坏稳定的“漩涡”的事件 DNA修复机制，这些中间产物然后通过危险的DNA修复途径进行处理， 仍未被开发。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)， 一种不同寻常的长链修复DNA合成，促进了遗传不稳定性的爆发；2) 微同源介导的BIR(MMBIR)，一种涉及多个模板切换事件的复制途径，位于 产生复杂基因组重排的微同源的位置；以及3)Long DSB的单链DNA中间体修复成“有毒”的联合分子，促进细胞死亡。作为一个开始 点，我们正在酵母中使用我们可靠和强大的系统，其中单个DSB由一个站点- 特异的HO内切酶；我们已经证明了所有三个感兴趣的修复事件都可以用于 修复这个系统中的损伤。使用这个系统获得的知识--修复机制， 中间体、参与蛋白质和突变模式-用于指导实验设计 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 各个项目的目标是相同的，并涉及三个主要的调查领域。首先，使用敏感基因 途径、蛋白质和DNA基序的存在影响修复中间产物进入 破坏稳定的修复机制的“漩涡”将被确定。第二，体内和体外的结合 将使用方法对单元的决策点进行建模和调查，以了解情况 (结构、动力学、参与蛋白质等)将中间体吸引到高风险和/或有毒修复中 小路。第三，由有害物质引起的突变和染色体重排的模式 将对修复路径进行评估，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，根据以前的研究开发的新软件MMBIRFinder将 用于检测当前可用的算法无法发现的复杂遗传变化。总体而言，这 研究计划将提高DNA修复中间处理机制的清晰度， 这将揭示影响危险修复路径调节的因素，并导致 真核生物基因组的不稳定。

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.