Visualization of break-induced replication.

描述(申请人提供)：生物不断受到诱变剂的攻击，包括来自各种来源的辐射和环境中的各种化学物质。细胞的存活依赖于对这些药物造成的DNA损伤的有效修复。这些药物引起的最致命的DNA损伤形式是双链断裂(DSB)，在没有DSB修复的情况下，细胞会迅速死亡。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，表征使细胞易受遗传不稳定性影响的途径对于理解肿瘤的发生至关重要，并可能有助于阐明癌症预防和治疗的靶点。这项拟议的研究将探讨断裂诱导复制(BIR)的机制，这是一种知之甚少的DSB修复途径，可导致遗传不稳定性。目前的BIR模型认为，入侵的断链和未受损的分子之间的连接启动了完整染色体上的DNA合成，从而作为复制的起点。这可能导致从供体分子复制数百千碱基的DNA，而在下一轮复制过程中，未修复的、断裂的DNA的一大部分会丢失。BIR被认为在修复倒塌的复制叉子中发挥重要作用，也在一些与癌症相关的现象中发挥作用，包括端粒在没有端粒酶的情况下维持、杂合性丧失和非互惠易位的形成。BIR与肿瘤发生机制的相关性使该模型成为许多分支研究的关键起点。然而，BIR-真诚复制修复DNA断裂的基本原理是基于遗传数据推导出来的，但从未通过对这一过程的中间产物和/或最终产物的物理分析直接证明。这项研究的目的是利用两种强大的技术，即动态分子梳理结合荧光原位杂交和双向凝胶电泳，来检验BIR的核心假设，即DSB修复是通过复制叉的组装和进展实现的。这项拟议的研究将确定酿酒酵母(一种模式遗传有机体)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位置进行复制的能力。此外，还将确定参与BIR的单个蛋白质的作用。这种方法是独特的，因为与其他仅依赖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来可视化单个DNA分子中的BIR，而二维凝胶电泳将有助于分析复制分叉中间体。这项研究旨在利用两种强大的技术：分子梳理和双向凝胶电泳法来确定断裂诱导复制(BIR)的机制。这项研究将测试BIR模型的基本原理，即组装一个真正的复制叉子来修复DNA的双链断裂。这些知识对于进一步理解一些与癌症相关的现象至关重要，这些现象包括端粒在没有端粒酶的情况下维持、杂合性丧失和非相互易位的形成。

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.