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）：生物体不断受到诱变剂的攻击，包括各种来源的辐射和环境中的各种化学品。细胞的存活取决于由这些试剂引起的DNA损伤的有效修复。由这些试剂诱导的DNA损伤的最致命形式是双链断裂（DSB），并且在没有DSB修复的情况下，细胞迅速死亡。然而，一些DSB修复途径是危险的，因为它们可能导致基因组不稳定，导致染色体重排。染色体重排与癌基因激活有关，是许多癌症的标志。因此，表征使细胞易于遗传不稳定的途径对于理解肿瘤发生至关重要，并可能有助于阐明癌症预防和治疗的靶点。这项研究将研究断裂诱导复制（BIR）的机制，这是一种知之甚少的DSB修复途径，可导致遗传不稳定。目前的BIR模型表明，入侵的断裂链和未受损分子之间的连接启动了完整染色体上的DNA合成，从而作为复制起点。这可能会导致从供体分子复制数百个DNA片段，而在下一轮复制过程中丢失了一大块未修复的断裂DNA。BIR已被认为在塌陷的复制叉的修复中起重要作用，并且还在几种癌症相关现象中起重要作用，包括在端粒酶不存在的情况下的端粒维持、杂合性丢失和非相互易位的形成。BIR与肿瘤发生机制的相关性使该模型成为许多研究分支的关键起点。然而，BIR的基本原则-真正的复制以修复DNA中的断裂是基于遗传数据推断的，但从未通过对该过程的中间体和/或最终产物的物理分析直接证明。拟议研究的目的是使用两种强大的技术，动态分子梳理与荧光原位杂交和二维凝胶电泳相结合，来测试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。拟议的研究将确定啤酒酵母（一种模式遗传生物）中BIR中间体和产物的结构。此外，它还将估计BIR的速度及其通过着丝粒和已知屏障部位复制的能力。此外，个别蛋白质参与BIR的作用将被确定。所提出的方法是独特的，因为与其他仅依赖于间接遗传或群体物理数据的BIR研究不同，它将使用动态分子梳理使单个DNA分子中的BIR可视化，而二维凝胶电泳将有助于分析复制叉中间体。本研究的目的是利用分子梳理和双向凝胶电泳两种强大的技术来确定断裂诱导复制（BIR）的机制。这项研究将测试BIR模型的基本原则，即组装一个真正的复制叉来修复DNA中的双链断裂。这些知识对于进一步理解几种癌症相关现象至关重要，包括在端粒酶不存在的情况下端粒的维持，杂合性丢失和非相互易位的形成。