Mechanisms of error prone repair of DNA breaks

摘要 微同源介导的末端连接(MMEJ)是一种进化保守的DNA双链修复途径 通过对小片段(2-20bps)重叠序列(微同源性；MH)进行退火链断裂(DSB) 在休息点的侧翼。按照设计，MMEJ是高度突变的，因为它总是导致一个基因的缺失 MH序列和MH间序列。MMEJ还经常导致染色体重排，原因是 它有能力进行混杂的末端连接。此外，我们发现MMEJ是高度诱变的，并且 从断裂开始，在修复连接两侧积累高达几千个碱基的突变。尽管如此 风险，MMEJ在细胞中被广泛采用，作为高保真DSB修复的替代选项 包括拷贝数变化、免疫系统发育和端粒融合在内的事件。最近， MMEJ也与致病的染色体易位有关，并正在成为一种有希望的 几种癌症的治疗靶点。总之，这些观察结果强调了 MMEJ是一种基因组不稳定因素，需要进一步研究以确定遗传和生化机制 MMEJ及其在各种环境和代谢条件下的调控。这样做的目的是 应用于解决有关MMEJ生理和病理作用的几个基本问题 所有真核细胞都有的。 MMEJ中尚未解决的关键问题之一是细胞如何识别和选择特定的MHS进行退火 以及MH中位置和错配次数对这一过程的影响。我们也不知道是什么 是催化MH退变的因素以及细胞如何调节修复选择以限制不想要的MMEJ和 抑制修复相关的染色体不稳定性。最后，我们将研究为什么老化会触发 修复途径的选择以及MMEJ活性如何导致酵母细胞中与年龄相关的染色体不稳定。 我们的初步结果已经揭开了几个有趣的MH退火规律和参数 确定了可能参与该反应的最终加工因素。我们还开发了一种新颖的记者 监测首次将MMEJ作为DSB修复选项之一的修复选择，并对其进行评估 DNA损伤反应和染色质重构体在决策过程中的作用。结果是 这项研究将揭示MMEJ关键步骤和功能相互作用的潜在机制 在DSB修复途径之间保持染色体的完整性以响应各种环境和 生理暗示。这些结果也将有助于确定MMEJ在许多基因组中的精确贡献 基因中随机序列的修饰事件和MMEJ产物的频率和类型预测 编辑和面临风险的基因组序列。

Abstract Microhomology-mediated end joining (MMEJ) is an evolutionary conserved pathway to repair DNA double strand breaks (DSBs) by annealing small stretches (2-20 bps) of overlapping sequence (microhomology; MH) flanking the break site. By design, MMEJ is highly mutagenic because it always results in the deletion of one of the MH and the inter-MH sequences. MMEJ also frequently leads to chromosomal rearrangements due to its ability to engage in promiscuous end joining. Moreover, we found that MMEJ is hypermutagenic, and accumulates mutations flanking the repair junctions up to several kilobases from the break. Despite these risks, MMEJ is widely adopted in cells as an alternative option to high fidelity DSB repair for a host of biological events including copy number variations, immune system development, and telomere fusions. Most recently, MMEJ has also been implicated in pathogenic chromosomal translocations and is emerging as a promising therapeutic target in several types of cancers. Together these observations underscore the importance of MMEJ as a genome destabilizer and warrant further studies to define genetic and biochemical mechanisms of MMEJ and its regulation under various environmental and metabolic conditions. The purpose of this application is to resolve several fundamental questions on the physiological and pathological roles of MMEJ common to all eukaryotic cells. One of the key unresolved questions in MMEJ is how cells recognize and select specific MHs for annealing and the effect of position and the number of mismatches in MH on this process. We also do not know what are the factors catalyzing MH annealing and how cells regulate repair choice to limit unwanted MMEJ and suppress repair-associated chromosomal instability. Lastly, we will examine why aging triggers changes in repair pathway choice and how MMEJ activity contributes to age related chromosomal instability in yeast cells. Our preliminary results already unraveled several interesting rules and parameters in MH annealing and identified end processing factors that are likely involved in this reaction. We also developed a novel reporter to monitor repair choice that for the first time includes MMEJ as one of the DSB repair options, and to evaluate the roles of DNA damage response and chromatin remodelers in the decision making process. The outcomes of this investigation will reveal the underlying mechanism of key steps in MMEJ and functional interactions between DSB repair pathways to sustain chromosomal integrity in response to various environmental and physiological cues. The results will also help establish precise contributions of MMEJ in many genome modification events and predict the frequency and types of MMEJ products at random sequences in gene editing and at-risk genome sequences.