CHEMICAL CARCINOGENESIS AND CELL PROLIFERATION

本项目的目标是描述 致癌物对细胞核DNA的损伤 在细胞周期的S期DNA复制。 我们有 观察到与复制叉相关的DNA 优先被化学致癌物甲基化，这表明 DNA复制可能影响致癌物的分布 与DNA结合。 我们现在建议确定这是否 现象可以在以下水平上观察和量化： 个体基因 在抗体的帮助下， 特异性DNA加合物，我们将沉淀DNA片段 包含病变，并确定该DNA部分是否是 富含在人类进化时期复制的 致癌物治疗 DNA中致癌物加合物的抗体， 以及与单链DNA区域结合的蛋白质， 将与电子显微镜一起使用， 加合物的分布和子链间隙的存在 DNA复制叉 我们将比较这些分布 与DNA合成抑制的生化模式， 致癌物治疗和致癌物对 同步化细胞中活性复制子的分布，如图所示 通过纤维放射自显影。 这种方法将提供新的见解 DNA链延长的机制，绕过DNA损伤 并从DNA复制的整体抑制中恢复。 的 DNA复制抑制的生化表征 将依赖于对新生DNA分子分布的研究 在碱性蔗糖梯度中， 异步的以及同步的小区。 后者将 在研究成熟过程中特别有用， 受损S期DNA复制中间产物的连接 细胞（即，复制后修复）。 的结果予以 实验将结合数据进行分析， 化学致癌物对细胞周期参数的影响， 例如细胞进入S期和M期的速率以及细胞周期的延长 复制期的一部分

The objective of this project is to characterize the relationship between carcinogen damage to nuclear DNA and the process of DNA replication during the S phase of the cell cycle. We have observed that DNA associated with replication forks is preferentially methylated by chemical carcinogens, suggesting that DNA replication may affect the distribution of carcinogen bound to DNA. We now propose to determine whether this phenomenon can be observed and quantitated at the level of individual genes. With the help of antibodies that recognize specific DNA adducts, we will precipitate DNA fragments containing lesions and determine whether this DNA fraction is enriched in gene sequences that were replicating at the time of carcinogen treatment. Antibodies to carcinogen adducts in DNA, together with proteins that bind to single-stranded DNA regions, will be used with the electron microscope to determine the distribution of adducts and the presence of daughter strand gaps at DNA replication forks. We will compare such distributions with the biochemical pattern of inhibition of DNA synthesis after carcinogen treatment and the effects of carcinogens on the distribution of active replicons in synchronized cells, as visualized by fiber autoradiography. Such approach will provide new insights into mechanisms of DNA chain elongation, by-pass of DNA lesions and recovery from the overall inhibition of DNA replication. The biochemical characterization of the inhibition of DNA replication will rely on the study of distributions of nascent DNA molecules in alkaline sucrose gradients following carcinogen treatment of asynchronous, as well as synchronized cells. The latter will be particularly useful in studying the process of maturation and joining of intermediates of DNA replication in damaged S phase cells (i.e., post-replication repair). The results of these experiments will be analyzed in conjunction with data on the effects of the chemical carcinogens on cell cycle parameters, such as rate of entry of cells into S and M phases and prolongation of the replicative period.