REPLICON MISFUNCTIONS IN HUMAN CARCINOGENESIS

描述：破坏DNA的致癌物质可能是暂时的或稳定的 扰乱个体高度有组织的DNA复制过程 复制并导致突变。该应用程序的目标是 人类细胞中分子因子的识别和理解 参与环肉烷胸腺嘧啶二聚体的旁路复制(T=T)。 无细胞萃取物中的DNA复制将在定义的附体上进行分析 含有唯一的T=T二聚体的DNA。描述了两个有重点的具体目标 来实现这些目标。第一个目标是确定分子 肿瘤易感人群中的嘧啶二聚体旁路复制缺陷 遗传性色素性干皮病(XPV)。试验性的 复制旁路缺陷的体外互补策略 XPV无细胞提取物使用纯化的复制/修复因子或 将开发HeLa细胞的分级提取物。使用基于SV40的 含有紫外线诱导的位点和链特异性的复制系统 曾用于沃斯·S博士实验室搭桥术的嘧啶二聚体 将评估主要DNA上的嘧啶二聚体的DNA合成 使用四个不同的XPV细胞系(两个EBV转化的 复制转化的淋巴母细胞和两株成纤维细胞系 SV40有缺陷)。第二个具体目标集中在分析 易于错误和无错误的旁路DNA复制对DNA的贡献 对人体无细胞萃取物的损害。诱变剂的贡献 跨损伤合成和同源链交换依赖的无错误 将确定嘧啶二聚体的旁路复制。它是 假设如果前导链T=T的无差错旁路复制 发生，那么这种DNA合成很可能涉及序列信息 来自另一条未受损的DNA链。这项工作将在#年进行 分两个阶段。首先，将开发一种分析方法来区分容易出错的 和位于T=T损伤部位的无错误DNA合成 领先的DNA链。其次，容易出错的和容易出错的 将评估旁路过程中的无错误复制，并将其用于隔离 绕过在各种细胞系中可能缺乏的因子。

DESCRIPTION: Carcinogenic agents that damage DNA may transiently or stably disrupt the highly organized process of DNA replication at individual replicons and lead to mutations. The goals of this application center on the identifying and understanding the molecular factors in human cells involved in bypass replication of cyclobotane thymine-thymine dimers (T=T). DNA replication in cell-free extracts will be analyzed on defined episomal DNA containing a unique T=T dimer. Two focused specific aims were described to achieve these objectives. The first aim seeks to determine the molecular defect in bypass replication of pyrimidine dimers in the cancer-prone hereditary disease xeroderma pigmentosum variant (XPV). An experimental strategy involving in vitro complementation of replication bypass deficient XPV cell-free extracts using purified replication/repair factors or fractionated extracts from HeLa cells will be developed. Using a SV40 based replication system containing a site- and strand-specific UV-induced pyrimidine dimer that was previously utilized in Dr. Vos s laboratory bypass DNA synthesis will be assessed for pyrimidine dimers on the leading DNA strand using four different XPV cell lines (two EBV-transformed lymphoblastoid and two fibroblast lines transformed with replication defective SV40). The second specific aim focuses on analyzing the contribution of error-prone and error-free bypass DNA replication of DNA damage in human cell-free extracts. The contribution of mutagenic translesion synthesis and homologous strand exchange-dependent error-free bypass replication of pyrimidine dimers will be determined. It is hypothesized that if error-free bypass replication of a leading strand T=T occurs, then such DNA synthesis most likely involves sequence information from the opposite undamaged DNA strand. This effort will be conducted in two phases. First, an assay will be developed to distinguish error-prone and error-free DNA synthesis at a site-specific T=T lesion located on the leading DNA strand. Second, the relative contribution of error-prone and error-free replication during bypass will be assessed and used to isolate bypass factors that may be deficient in various cell lines.