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: 8758960
• 负责人: Anna L Malkova
• 金额: $ 29.92万
• 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/8758960
• 关键词: Biological Assay; Cancer Biology; Cancerous; Chromosomal Rearrangement; Chromosomes; Comb animal structure; Complex; Copy Number Polymorphism; Coupled; DNA; DNA Sequence; DNA Sequence Rearrangement; 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; 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; data mining; expectation; fight against; genetic analysis; migration; molecular dynamics; new technology; novel; prevent; public health relevance; 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）：遗传不稳定性在癌变中起着至关重要的作用，使有关导致基因组重排和诱变的机制的知识是对抗癌症的关键工具。该项目的重点是一种新型的DNA合成类型，迁移的bubble DNA合成（MIBS），它促进了基因组不稳定性的爆发，包括超氧化，易位，易位和拷贝数变化。与S期复制形成鲜明对比的是，MIB是在双链断裂（DSB）位点而不是在复制原点上启动的，由迁移的气泡而不是通过复制叉进行，并导致新合成DNA的保守遗传。该建议旨在揭示MIB的分子机制，并确定MIB如何促进人类癌症的各种遗传不稳定性。为了研究MIB，我们将在酿酒酵母的酵母中使用可靠且功能强大的系统，在酵母中，由特定地点的HO内核酸内切酶引发的单个DSB通过断裂诱导的复制（BIR）修复，这是一个重要的DSB修复途径，该途径通过MIBS进行。更具体地说，DSB通过侵袭一个断裂的DNA的一个自由端进入同源染色体，然后由MIBS介导的DNA合成，该DNA合成由MIBS介导，该MIB介导了大约100千座到同源物的末端，从而导致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MIB的机制，并表征负责其负责的复制蛋白的作用。我们将进一步确定MIB促进的诱变增加的机制，采用敏感的遗传分析来充分表征DNA聚合酶在MIBS相关的高渗透性中的作用，并评估MIB在使用全基因组DNA测序的突变簇形成中的作用。重要的是，这些研究的结果将揭示出区域性超粘性的机制，即卡特吉斯（Kataegis），最近在各种类型的癌症中进行了描述。最后，我们将确定MIB在促进与与染色体相似的复合GCR中的作用，与染色体相似，这是一种与癌症相关的现象，涉及局限于单个染色体的大规模基因组变化。在PI的实验室中获得的初步结果表明，当DSB修复从MIBS转换为微型学介导的BIR（MMBIR）时，可能会发生类似Chromothripsis的GCR。拟议的研究将揭示从MIBS到MMBIR的介导的转换的机理，包括跨DNA聚合酶在此过程中的作用，并确定MMBIR在GCR形成中的作用。总体而言，这项提出的研究的结果有望建立一个新的概念：这样的观念是，可能导致癌症的一系列遗传不稳定可能是由于不寻常的复制（MIB）而导致的，而不是由于半稳态S相复制期间遗传变化的较小遗传变化的持续积累。