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

双链断裂修复路线错误导致风险放大

• 批准号: 8892195
• 负责人: Anna L Malkova
• 金额: $ 28.72万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-07-01 至 2018-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8892195
• 关键词: Biological Assay; Cancer Biology; Cancerous; Chromosomal Rearrangement; Chromosomes; Comb animal structure; Complex; Copy Number Polymorphism; Coupled; DNA; DNA Sequence; DNA biosynthesis; DNA-Directed DNA Polymerase; Development; Disease; Double Strand Break Repair; Electron Microscopy; Event; Fluorescent in Situ Hybridization; Gene Conversion; Genetic; Genetic Materials; Genome; Genomic Instability; Genomics; Goals; HO nuclease; Health; Homologous Gene; Human; Human Characteristics; Interruption; Investigation; Knowledge; Lead; Left; Lesion; Light; Malignant Neoplasms; Mammals; Mediating; Methods; Modeling; Modification; Molecular; Mutagenesis; Mutation; Outcome; Pathway interactions; Pattern; Play; Polymerase; Process; Property; Protein Biosynthesis; Proteins; Replication Error; Replication Origin; Research; Risk; Role; S Phase; Saccharomyces cerevisiae; Single-Stranded DNA; Site; Structure; System; Testing; Two-Dimensional Gel Electrophoresis; Work; Yeasts; cancer cell; cancer genome; cancer type; carcinogenesis; chromothripsis; data mining; expectation; fight against; genetic analysis; migration; molecular dynamics; new technology; novel; prevent; repaired; telomere; therapeutic target; tool; tumorigenesis; two-dimensional

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.

描述（由申请人提供）：遗传不稳定性在致癌作用中起着关键作用，使有关导致基因组重排和诱变的机制的知识成为对抗癌症的关键工具。该项目的重点是一种新型的DNA合成，迁移气泡DNA合成（MiBS），它促进了基因组不稳定性的爆发，包括超诱变，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）位点而不是在复制起点处起始，通过迁移气泡而不是通过复制叉进行，并且导致新合成的DNA的保守遗传。该提案旨在揭示MiBS的分子机制，并确定MiBS如何促进人类癌症特征的各种类型的遗传不稳定性。为了研究MiBS，我们将在酵母中使用可靠且强大的系统，酿酒酵母（Saccharomyces cerevisiae），其中由位点特异性HO内切核酸酶启动的单个DSB通过断裂诱导复制（BIR）修复，BIR是通过MiBS进行的重要DSB修复途径。更具体地说，通过将断裂DNA的一个游离端侵入同源染色体，然后通过MiBS介导的DNA合成来修复DSB，所述DNA合成进行约100个酶至同源物的末端，从而产生具有正常端粒的修复分子。我们将使用直接物理方法，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表征DNA聚合酶在MiBS相关的超变中的作用，并使用全基因组DNA测序评估MiBS在突变簇形成中的作用。重要的是，这些研究的结果将揭示区域超突变性的机制，kataegis，最近在各种类型的癌症中被描述。最后，我们将确定MiBS在促进复杂GCR中的作用，这些GCR类似于与chromothripsis相关的GCR，这是一种与癌症相关的现象，涉及定位于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，可能会发生色颤样GCR。拟议的研究将揭示从MiBS到MMBIR的介导转换机制，包括translesion 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，这项拟议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爆发可能是由一种不寻常的复制类型（MiBS）引起的，而不是由半保守S期复制期间小的遗传变化的持续积累引起的。