Thomas D C, Roberts J D, Fitzgerald M P, Kunkel T A
Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709.
Basic Life Sci. 1990;52:289-97. doi: 10.1007/978-1-4615-9561-8_24.
We are investigating the mechanisms by which mutations are produced or avoided during DNA synthesis. Using in vitro fidelity assays, we have defined the error frequency and mutational specificity of the replicative animal cell DNA polymerases (alpha and delta). With DNA polymerase alpha or the four-subunit DNA polymerase alpha-DNA primase complex, neither of which contains detectable associated exonuclease activity, the fidelity of the polymerization step is low relative to spontaneous mutation rates in vivo. DNA polymerase delta is much more accurate, partly due to proofreading by the 3'----5' exonuclease activity associated with this polymerase. These fidelity studies have been extended to the replication apparatus present in extracts of human HeLa cells. The replication complex is highly accurate, suggesting that additional fidelity components are operating in the extract during bidirectional, semiconservative replication of double-stranded DNA. Nevertheless, in highly sensitive reversion assays, base substitution errors can be readily detected at frequencies greater than the estimated rate of spontaneous mutation in vivo. This suggests that fidelity components may be missing and/or that human cells depend heavily on postreplicative repair processes to correct replication errors.
我们正在研究DNA合成过程中产生或避免突变的机制。通过体外保真度测定，我们已经确定了复制性动物细胞DNA聚合酶（α和δ）的错误频率和突变特异性。对于DNA聚合酶α或四亚基DNA聚合酶α-DNA引发酶复合物，这两者均不包含可检测到的相关外切核酸酶活性，相对于体内的自发突变率，聚合步骤的保真度较低。DNA聚合酶δ更为精确，部分原因是与该聚合酶相关的3'→5'外切核酸酶活性进行的校对。这些保真度研究已扩展至人HeLa细胞提取物中存在的复制装置。复制复合物高度精确，这表明在双链DNA的双向半保留复制过程中，提取物中存在其他保真度组分在起作用。然而，在高度灵敏的回复突变测定中，可以很容易地检测到碱基替换错误，其频率高于体内估计的自发突变率。这表明可能缺少保真度组分和/或人类细胞严重依赖复制后修复过程来纠正复制错误。
