Amplification of risk resulting from mis-routing of double-strand break repair

描述（由申请人提供）：遗传不稳定性在癌症发生中起着关键作用，使有关导致基因组重排和突变的机制的知识成为对抗癌症的关键工具。该项目专注于一种新型的DNA合成，即迁移泡DNA合成（MiBS），它促进了基因组不稳定性的爆发，包括超突变、易位和拷贝数变异。与S期复制形成鲜明对比的是，MiBS在双链断裂（DSB）位点而不是复制起点启动，由迁移泡而不是复制叉进行，并导致新合成DNA的保守遗传。本提案旨在揭示MiBS的分子机制，并确定MiBS如何促进人类癌症特征的各种遗传不稳定性。为了研究MiBS，我们将在酵母中使用一个可靠而强大的系统，酿酒酵母，其中由位点特异性HO内切酶启动的单个DSB通过断裂诱导复制（BIR）修复，这是通过MiBS进行的重要DSB修复途径。更具体地说，DSB的修复是通过将断裂DNA的一个自由端侵入同源染色体，然后由MiBS介导的DNA合成进行大约100千碱基的同源物末端，从而产生具有正常端粒的修复分子。我们将使用直接的物理方法，包括二维凝胶电泳、动态分子梳理和电子显微镜来确定MiBS的机制，并表征负责它的复制蛋白的作用。我们将进一步确定MiBS促进突变增加的机制，采用敏感的遗传分析来充分描述DNA聚合酶在MiBS相关的高易变性中的作用，并使用全基因组DNA测序来评估MiBS在突变簇形成中的作用。重要的是，这些研究结果将揭示最近在各种类型的癌症中描述的区域超易变性（kataegis）的机制。最后，我们将确定MiBS在促进与染色体断裂相关的复杂gcr中的作用，染色体断裂是一种与癌症相关的现象，涉及单个染色体的大量基因组变化。PI实验室获得的初步结果表明，当DSB修复从MiBS切换到微同源介导的BIR （MMBIR）时，可能会发生类似嗜色缺陷的gcr。本研究将揭示从MiBS到MMBIR的转换机制，包括翻译DNA聚合酶在这一过程中的作用，并将确定MMBIR在gcr形成中的作用。总的来说，这项研究的结果有望建立一个新的概念：可能导致癌症的遗传不稳定性的爆发可能是由一种不寻常的复制类型（MiBS）引起的，而不是在半保守的s期复制期间持续积累的小遗传变化。

DESCRIPTION (provided by applicant): Genetic instability plays a critical role in carcinogenesis, making knowledge about the mechanisms that lead to genome rearrangements and mutagenesis a critical tool in the fight against cancer. This project is focused on a novel type of DNA synthesis, migrating-bubble DNA synthesis (MiBS), which promotes bursts of genomic instability, including hyper-mutagenesis, translocations, and copy number variations. In stark contrast to S- phase replication, MiBS is initiated at a double-strand break (DSB) site rather than at a replication origin, is carried out by a migrating bubble rather than by a replication fork, and leads to conservative inheritance of newly synthesized DNA. This proposal aims to unravel the molecular mechanism of MiBS and to determine how MiBS promotes various types of genetic instabilities characteristic of human cancers. To study MiBS, we will use a dependable and powerful system in yeast, Saccharomyces cerevisiae, where a single DSB initiated by a site-specific HO endonuclease is repaired by break-induced replication (BIR), an important DSB repair pathway which proceeds through MiBS. More specifically, a DSB is repaired by invasion of one free end of broken DNA into the homologous chromosome followed by DNA synthesis mediated by MiBS that proceeds for approximately 100 kilobases to the end of the homologue, resulting in a repaired molecule with a normal telomere. We will use direct physical methods, including two-dimensional gel electrophoresis, dynamic molecular combing, and electron microscopy to determine the mechanism of MiBS and to characterize the roles of replication proteins that are responsible for it. We will further determine the mechanism of increased mutagenesis promoted by MiBS, employ sensitive genetic analyses to fully characterize the role of DNA polymerases in MiBS-associated hypermutability, and assess the role of MiBS in the formation of mutation clusters using whole-genome DNA sequencing. Importantly, the results of these investigations will shed light on a mechanism of regional hyper-mutability, kataegis, which has recently been described in various types of cancer. Finally, we will determine the role of MiBS in promoting complex GCRs similar to those associated with chromothripsis, a cancer-related phenomenon that involves massive genomic changes localized to a single chromosome. Preliminary results obtained in the PI's lab suggest that chromothripsis-like GCRs may occur when DSB repair switches from MiBS to microhomology-mediated BIR (MMBIR). The proposed research will unravel the mechanism mediating switches from MiBS to MMBIR, including the role of translesion DNA polymerases in this process, and will determine the role of MMBIR in formation of GCRs. Overall, the results of this proposed research are expected to establish a novel concept: the notion that a burst of genetic instabilities that can lead to cancer may result from an unusual type of replication (MiBS) rather than from a continuing accumulation of small genetic changes during semi-conservative S-phase replication.