Visualization of break-induced replication.

断裂诱导复制的可视化。

• 批准号: 7661619
• 负责人: KIRILL S LOBACHEV
• 金额: $ 7.54万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-07-21 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7661619
• 关键词: Biological Phenomena; Cell Survival; Cells; Centromere; Chemicals; Chromosomal Rearrangement; Chromosomes; Collaborations; Comb animal structure; Communities; Coupled; DNA; DNA Damage; DNA Double Strand Break; DNA Repair; DNA biosynthesis; DNA replication origin; Data; Development; Double Strand Break Repair; Environment; Exposure to; Fluorescent in Situ Hybridization; Funding; Gene Conversion; Genetic; Genetic Models; Genetic Recombination; Genomic Instability; Grant; Hand; Hereditary Disease; Homeostasis; Human Pathology; Imagery; Indiana; Individual; Institutes; Invaded; Knowledge; Laboratories; Lead; Life; Loss of Heterozygosity; Malignant Neoplasms; Modeling; Molecular; Monitor; Nature; Oncogene Activation; Organism; Outcome; Pathway interactions; Play; Population; Process; Proteins; Radiation; Reciprocal Translocation; Replication Origin; Research; Research Project Grants; Role; Saccharomyces cerevisiae; Site; Source; Speed; Structure; Techniques; Technology; Telomerase; Telomere Maintenance; Testing; Two-Dimensional Gel Electrophoresis; Universities; Yeasts; assault; base; cancer prevention; design; dimer; environmental agent; innovation; molecular dynamics; mutant; recombinational repair; repaired; telomere; tumorigenesis; two-dimensional; yeast protein

DESCRIPTION (provided by applicant): Living organisms are under constant assault by mutagenic agents, including radiation from various sources and a wide range of chemicals in the environment. Cell survival is dependent upon effective repair of DNA damage caused by these agents. The most lethal form of DNA damage induced by these agents is double-strand breaks (DSBs) and, in the absence of DSB repair, cells die rapidly. However, some DSB repair pathways are dangerous because they can lead to genomic instability, resulting in chromosomal rearrangements. Chromosomal rearrangements have been implicated in oncogene activation and are the hallmark of many cancers. Therefore, characterization of pathways that predispose cells to genetic instability is critical for the understanding of tumorigenesis and may help to elucidate targets for cancer prevention and treatment. The proposed research will investigate the mechanism of break-induced replication (BIR), a poorly understood DSB repair pathway, which can lead to genetic instability. The current BIR model suggests that the junction made between the invading broken strand and the undamaged molecule initiates DNA synthesis on the intact chromosome, thereby acting as an origin of replication. This can result in copying of hundreds of kilobases of DNA from the donor molecule while a large piece of the unrepaired, broken DNA is lost during the next round of replication. BIR has been suggested to play an important role in the repair of collapsed replication forks, and also in several cancer-related phenomena, including telomere maintenance in the absence of telomerase, loss of heterozygosity, and formation of non-reciprocal translocations. The relevance of BIR to mechanisms underlying tumorigenesis has made this model a critical starting point for many branches of research. However, the basic tenet of BIR - bona fide replication to repair breaks in DNA was deduced based on genetic data but has never been demonstrated directly by physical analyses of intermediates and/or the final products of this process. The objective of the proposed research is to use two powerful technologies, dynamic molecular combing coupled with fluorescent in situ hybridization and two- dimensional gel electrophoresis, to test the central hypothesis of BIR; i.e., that DSB repair is achieved by the assembly and progression of a replication fork. The proposed research will determine the structure of BIR intermediates and products in yeast Saccharomyces cerevisiae (a model genetic organism). Also, it will estimate the speed of BIR and its ability to replicate through centromeres and known barrier sites. In addition, the roles of individual proteins involved in BIR will be identified. The proposed approach is unique because, unlike other studies of BIR that rely only on indirect genetic or population physical data, it will enable visualization of BIR in individual DNA molecules using dynamic molecular combing, while two-dimensional gel electrophoresis will help to analyze replication fork intermediates.Narrative. The proposed research is aimed to determine the mechanism of break-induced replication (BIR) by using two powerful technologies: molecular combing and two-dimensional gel electrophoresis. This research will test the basic tenet of the BIR model, namely, the assembly of a bona fide replication fork to repair double-strand breaks in DNA. This knowledge is critical to further the understanding of several cancer-related phenomena, including telomere maintenance in the absence of telomerase, loss of heterozygosity, and formation of non-reciprocal translocations.

描述（由申请人提供）：活生物体受到诱变剂的持续攻击，包括来自各种来源的辐射和环境中各种化学物质。细胞存活取决于这些药物引起的有效修复DNA损伤。这些药物引起的DNA损伤的最致命形式是双链断裂（DSB），在没有DSB修复的情况下，细胞迅速死亡。但是，某些DSB修复途径很危险，因为它们可能导致基因组不稳定性，从而导致染色体重排。染色体重排与癌基因的激活有关，并且是许多癌症的标志。因此，表征使遗传不稳定性易感细胞的途径对于理解肿瘤发生至关重要，并且可能有助于阐明预防癌症和治疗的靶标。拟议的研究将研究突破性诱导的复制（BIR）的机制，这是DSB修复途径知之甚少，这可能导致遗传不稳定。当前的BIR模型表明，入侵的链链和未损坏的分子之间产生的连接启动了完整染色体的DNA合成，从而充当复制的起源。这可能导致从供体分子中复制数百千碱基的DNA，而在下一轮复制中，一大块未修复的，断裂的DNA丢失了。已经建议BIR在修复崩溃的复制叉的修复以及几种与癌症相关的现象中发挥重要作用，包括在没有端粒酶的情况下维持端粒的维持，杂合性丧失以及形成非互联体易位。 BIR与肿瘤发生的机制的相关性使该模型成为许多研究分支的关键起点。但是，根据遗传数据推导了BIR -BIR的基本宗旨 - 对DNA中修复休息的复制，但从未通过中间体和/或该过程的最终产物的物理分析直接证明。拟议的研究的目的是使用两种强大的技术，即动态分子梳理，并结合荧光原位杂交和两维凝胶电泳，以测试BIR的中心假设。即，DSB修复是通过复制叉的组装和进展来实现的。拟议的研究将确定酿酒酵母（一种模型遗传生物）中BIR中间体和产物的结构。同样，它将估计BIR的速度及其通过centromeres和已知屏障站点复制的能力。此外，将确定与BIR有关的个别蛋白质的作用。所提出的方法是独一无二的，因为与其他仅依赖间接遗传或种群物理数据的BIR研究不同，它将使用动态分子梳理能够在单个DNA分子中可视化BIR，而二维凝胶电泳将有助于分析复制fork Intermediates。拟议的研究旨在通过使用两种强大的技术来确定破裂诱导的复制（BIR）的机制：分子梳和二维凝胶电泳。这项研究将测试BIR模型的基本原则，即，善意复制叉的组装以修复DNA中的双链断裂。这些知识对于进一步了解几种与癌症相关现象的理解至关重要，包括在没有端粒酶，杂合性丧失以及非逆转录易位的形成的情况下维持端粒。