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

DNA损伤的准确修复对细胞的生存和维持基因组的稳定至关重要。 双链DNA断裂(DSB)是最致命的DNA损伤，细胞已经进化出各种 它们的修复机制。虽然一些DSB修复路径是准确的，但其他路径可能会破坏 通过产生与癌症和其他人类疾病相关的突变或染色体重排来获得基因组 疾病。我们的长期目标是找出驱动dsb修复进入有害dna漩涡的因素。 修复途径，并表征其分子机制。我们专注于两个这样的高风险DSB修复 途径：1)断裂诱导复制(BIR)，一种不寻常的长链修复DNA合成，促进 遗传不稳定性的爆发；以及2)微同源介导的BIR(MMBIR)，一种复制途径，涉及 在产生复杂基因组的微同源物位置上的多个模板切换事件 重新安排。我们将使用一种具有广泛特征的、强大的酵母系统来研究遗址的修复- 特定的HO-内切酶诱导的DSB为其他系统中的研究设计提供信息。Mira支持 使我们在BIR和MMBIR的表征方面取得了重大进展，包括开发了几个 创新工具。其中之一，我们将其命名为琥珀(用于监测BIR延伸率的分析)，是 基于液滴数字聚合酶链式反应的方法，以前所未有的分辨率测量BIR动力学。使用琥珀色 在下一个Mira支持周期内，我们将能够确定由我们的 新发现的BIR驱动蛋白候选，包括纺锤体组装检查点蛋白。我们还将使用 我们敏感的酵母BIR系统中的琥珀色，以解开碰撞后BIR调节的机制 具有各种复制障碍，包括表征依赖于Rad52的单链的作用 为碰撞后的BIR重新启动进行退火热处理。所得结果将为阐明RAD51的作用机制奠定基础。 酵母中独立的BIR，这是一个很可能类似于哺乳动物中描述的BIR事件的途径。 我们在MIRA支持下开发的另一种方法支持基于Long的BIR事件检测 在APOBEC(胞苷脱氨酶)存在下，由BIR形成的突变簇，我们建议 将此方法应用于检测酵母减数分裂过程中的BIR。确定诱变BIR的频率 可能用于修复减数分裂DSB是重要的，因为类似的事件可能会导致人类的出生缺陷。 最后，我们的新软件MMBSearch-基于我们对酵母中MMBIR的表征开发的-将是 用于识别使人类细胞易受MMBIR事件影响的特定条件，我们最近发现 在癌症中很常见，但在非癌症细胞中很少见。MMBSearch在全基因组测序中的应用 数据将确定特定的癌症、细胞类型、染色体位置和促进 MMBIR。总体而言，这项研究计划将产生关于促进风险的因素的基础知识 DSB修复途径以及这些途径可能破坏真核基因组稳定的机制。

Accurate repair of DNA lesions is paramount to the survival of cells and to maintain their genomic stability. Double-strand DNA breaks (DSBs) are the most lethal DNA lesion, and cells have evolved a variety of mechanisms for their repair. While some DSB repair pathways are accurate, others can destabilize the genome by creating mutations or chromosome rearrangements associated with cancer and other human diseases. Our long-term goal is to identify factors that drive DSB repair into the maelstrom of deleterious DNA repair pathways, and to characterize their molecular mechanisms. We focus on two such high-risk DSB repair pathways: 1) break-induced replication (BIR), an unusual type of long-tract repair DNA synthesis that promotes bursts of genetic instabilities; and 2) microhomology-mediated BIR (MMBIR), a replicative pathway involving multiple template-switching events at positions of microhomologies that yields complex genomic rearrangements. We will use an extensively characterized, powerful yeast system to study repair of a site- specific HO-endonuclease-induced DSB to inform the design of studies in other systems. MIRA support enabled significant progress in our characterization of BIR and MMBIR, including development of several innovative tools. One of them, which we named AMBER (Assay for Monitoring BIR Elongation Rate), is a droplet-digital-PCR-based method to measure BIR kinetics with unprecedented resolution. Using AMBER during the next MIRA support cycle will allow us to identify the specific steps of BIR that are controlled by our newly identified BIR driver protein candidates, including spindle assembly checkpoint proteins. We will also use AMBER in our sensitive yeast BIR system to unravel the mechanisms of BIR regulation following its collision with various replication obstacles, including characterizing the role of Rad52-dependent single-strand annealing for BIR re-start after collision. The obtained results will shed light on the mechanism of Rad51- independent BIR in yeast, which is a pathway that is likely similar to BIR events described in mammals. Another approach that we developed with MIRA support enabled the detection of BIR events based on long mutation clusters formed by BIR occurring in the presence of APOBEC (cytidine deaminase), and we propose to apply this methodology here to detect BIR during yeast meiosis. Determining how frequently mutagenic BIR might be used to repair meiotic DSBs is important because similar events can lead to birth defects in humans. Finally, our new software, MMBSearch—developed based on our characterization of MMBIR in yeast—will be used to identify specific conditions that predispose human cells to MMBIR events, which we recently found to be frequent in cancer, but rare in non-cancerous cells. Applying MMBSearch to whole-genome sequencing data will identify specific cancers, cell types, chromosomal locations and environmental stressors that promote MMBIR. Overall, this research program will produce fundamental knowledge on the factors that promote risky DSB repair pathways and the mechanisms of these pathways that can destabilize eukaryotic genomes.