REPLICON MISFUNCTIONS IN HUMAN CARCINOGENESIS

描述：致癌剂损伤DNA可能是短暂或稳定的 破坏个体高度有序的DNA复制过程， 复制子并导致突变。 此应用程序的目标集中在 识别和理解人类细胞中的分子因子 参与环己基胸腺嘧啶-胸腺嘧啶二聚体（T = T）的旁路复制。 无细胞提取物中的DNA复制将在定义的附加体上进行分析。 含有独特的T = T二聚体的DNA。 描述了两个有重点的具体目标 以实现这些目标。 第一个目标是确定分子 癌易感细胞中嘧啶二聚体旁路复制缺陷 遗传性着色性干皮病变异型（XPV）。 实验 涉及体外互补复制旁路缺陷的策略 使用纯化的复制/修复因子的XPV无细胞提取物，或 将开发来自HeLa细胞的分级提取物。 基于SV40 复制系统包含位点和链特异性紫外线诱导的 嘧啶二聚体，以前用于Vos博士的实验室旁路 DNA合成将评估前导DNA上的嘧啶二聚体 使用四种不同的XPV细胞系（两种EBV转化的 淋巴母细胞和两个复制转化的成纤维细胞系 有缺陷的SV40）。 第二个具体目标是分析 易错和无错旁路DNA复制的贡献 人体无细胞提取物中的损伤。 致突变的贡献 跨损伤合成和同源链交换依赖的无错 测定嘧啶二聚体的旁路复制。 是 假设如果前导链T = T的无错误旁路复制 发生，那么这种DNA合成很可能涉及序列信息， 从另一条未受损的DNA链中分离出来 这项工作将在 两个阶段。 首先，将开发一种检测方法来区分易出错的 和位于细胞核上的位点特异性T = T损伤处的无错误DNA合成。 前导DNA链 第二，易错和 将评估旁路期间的无错误复制并用于隔离 旁路因子可能在各种细胞系中缺乏。

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.