ACCURACY OF DNA REPLICATION IN VITRO

这个项目的目标是了解三个过程负责 人类细胞中DNA复制保真度：DNA聚合酶基础 选择性、核酸外切校正和复制后错配 修复. 我们已经确定了DNA合成的保真度， 真核生物的五种DNA聚合酶。 DNA聚合酶具有 明显不同的错误率和特异性， 它们在DNA复制的各个阶段中的作用。 我们还确定了双向DNA复制的保真度， 正常人细胞提取物中的多蛋白复制装置 和肿瘤细胞。 尽管复制的总体保真度与 在两条链上，碱基替换和移码错误率确实 在前导链和滞后链复制的某些位点上不同 设备. 为了更好地了解已知诱变剂的影响 和致癌物质对DNA合成的保真度，我们正在执行 含DNA的DNA分子复制保真度的研究 加合物现在的重点是在适当放置的AAF加合物，定义 终止与旁路的概率和致突变程度 旁路。 我们已经证明，在细胞中的多蛋白复制装置 人HeLa细胞的提取物确实能够致突变translesion 绕过了三种非常不同的加合物，这意味着 在癌症病因学中的作用。 以试图 了解某些肿瘤中微卫星序列的不稳定性 以及几种遗传性疾病中三联体重复序列的不稳定性， 人类疾病，我们最近开始研究的fidelity 简单重复的二核苷酸和三核苷酸重复序列的复制。 最后，我们正在研究正常和突变的人类细胞 用于校正含有错配或未配对的DNA底物的提取物 个核苷酸 这些研究对于了解分子生物学 致癌起始事件的遗传基础和 暴露在DNA损伤的环境中， 剂.

The goal of this project is to understand the three processes reponsible for DNA replication fidelity in human cells: DNA polymerase base selectivity, exonucleolytic proofreading and post-replication mismatch repair. We have determined the fidelity of DNA synthesis catalyzed by the five classes of eukaryotic DNA polymerases. The DNA polymerases have distinctly different error rates and specificities, which has implications for their roles in the various stages of DNA replication. We have also determined the fidelity of bidirectional DNA replication by the multiprotein replication apparatus in extracts of normal human cells and tumor cells. Although the overall fidelity of replication is similar on the two strands, base substitution and frameshift error rates do differ at some sites for the leading and lagging strand replication apparatus. In order to better understand the effects of known mutagens and carcinogens on the fidelity of DNA synthesis, we are performing studies of replication fidelity with DNA molecules containing DNA adducts. Emphasis is now on uniquely-placed AAF adducts, defining the probability of termination versus bypass and the extent of mutatgenic bypass. We have shown that the multiprotein replication apparatus in extracts of human HeLa cells is indeed capable of mutagenic translesion bypass of three very different types of adducts, which has implications for their possible roles in the etiology of cancer. In an attempt to understand the instabilty of microsatellite sequences in certain tumors and the instability in triplet repeat sequences in several hereditary human diseases, we have recently begun to examine the fideilty of replication of simple repetitive di- and tri-nucleotide repeat sequences. Lastly, we are examining the ability of normal and mutant human cell extracts to correct DNA substrates containing mispaired or unpaired nucleotides. These studies are important for understanding the molecular genetic basis for the initiating events in carcinogenesis and the risk posed to individuals in the population by exposure to DNA damaging agents.