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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

基本信息

批准号：
9280976
负责人：
Anna L Malkova
金额：
$ 28.46万
依托单位：
UNIVERSITY OF IOWA
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-07-01 至 2019-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9280976
关键词：
Biological Assay Cancer Biology Chromosomal Rearrangement Chromosomes Comb animal structure Complex Copy Number Polymorphism Coupled DNA DNA biosynthesis DNA replication fork DNA sequencing 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 Premalignant Premalignant Cell Process Property Protein Biosynthesis Proteins Recruitment Activity Replication Error Replication Origin Research Risk Role Route 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 gel electrophoresis genetic analysis migration molecular dynamics new technology novel prevent public health relevance repaired telomere therapeutic target tool tumorigenesis two-dimensional whole genome

项目摘要

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过程中起着至关重要的作用，使得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。该项目专注于一种新型 DNA 合成，即迁移泡 DNA 合成 (MiBS)，它会促进基因组不稳定的爆发，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起始，通过迁移气泡而不是复制叉进行，并导致新合成 DNA 的保守遗传。该提案旨在揭示 MiBS 的分子机制，并确定 MiBS 如何促进人类癌症的各种类型的遗传不稳定性特征。为了研究 MiBS，我们将在酿酒酵母中使用可靠而强大的系统，其中由位点特异性 H2O 核酸内切酶启动的单个 DSB 通过断裂诱导复制 (BIR) 进行修复，BIR 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 的一个游离端侵入同源染色体，然后由 MiBS 介导进行 DNA 合成，合成大约 100 KB 至同源体末端，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，并表征负责其的复制蛋白的作用。我们将进一步确定 MiBS 促进突变增加的机制，采用灵敏的遗传分析来充分表征 DNA 聚合酶在 MiBS 相关超突变中的作用，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机制 kataegis，最近在各种类型的癌症中描述了这种机制。最后，我们将确定 MiBS 在促进与染色体碎裂相关的复杂 GCR 中的作用，染色体碎裂是一种与癌症相关的现象，涉及局限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，可能会发生类似染色体碎裂的 GCR。拟议的研究将揭示从 MiBS 到 MMBIR 的介导机制，包括跨损伤 DNA 聚合酶在此过程中的作用，并将确定 MMBIR 在 GCR 形成中的作用。总的来说，这项研究的结果预计将建立一个新的概念：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。

项目成果

期刊论文数量（11）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Cascades of genetic instability resulting from compromised break-induced replication.

DOI：
10.1371/journal.pgen.1004119
发表时间：
2014-02
期刊：
PLoS genetics
影响因子：
4.5
作者：
Vasan S;Deem A;Ramakrishnan S;Argueso JL;Malkova A
通讯作者：
Malkova A

Break-induced replication is highly inaccurate.

DOI：
10.1371/journal.pbio.1000594
发表时间：
2011-02-15
期刊：
PLoS biology
影响因子：
9.8
作者：
Deem A;Keszthelyi A;Blackgrove T;Vayl A;Coffey B;Mathur R;Chabes A;Malkova A
通讯作者：
Malkova A

Microhomology directs diverse DNA break repair pathways and chromosomal translocations.