Thomas D C, Roberts J D, Kunkel T A
Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709.
J Biol Chem. 1991 Feb 25;266(6):3744-51.
A general repair process for DNA heteroduplexes has been detected in HeLa cell extracts. Using a variety of M13mp2 DNA substrates containing single-base mismatches and extra nucleotides, extensive repair is observed after incubation with HeLa cell cytoplasmic extracts and subsequent transfection of bacterial cells with the treated DNA. Most, but not all, mispairs as well as two frameshift heteroduplexes are repaired efficiently. Parallel measurements of repair in HeLa extracts and in Escherichia coli suggest that repair specificities are similar for the two systems. The presence of a nick in the molecule is required for efficient repair in HeLa cell extracts, and the strand containing the nick is the predominantly repaired strand. Mismatch-dependent DNA synthesis is observed when radiolabeled restriction fragments, produced by reaction of the extract with heteroduplex and homoduplex molecules, are compared. Specific labeling of fragments, representing a region of approximately 1,000 base pairs and containing the nick and the mismatch, is detected for the heteroduplex substrate but not the homoduplex. The repair reaction is complete after 20 min and requires added Mg2+, ATP, and an ATP-regenerating system, but not dNTPs, which are present at sufficient levels in the extract. An inhibitor of DNA polymerase beta, dideoxythimidine 5'-triphosphate, does not inhibit mismatch-specific DNA synthesis. Aphidicolin, an inhibitor of DNA polymerases alpha, delta, and epsilom, inhibits both semiconservative replication and repair synthesis in the extract. Butylphenyl-dGTP also inhibits both replicative and repair synthesis but at a concentration known to inhibit DNA polymerase alpha preferentially rather than delta or epsilon. This suggests that DNA polymerase alpha may function in mismatch repair.
在HeLa细胞提取物中检测到了一种DNA异源双链体的一般修复过程。使用多种含有单碱基错配和额外核苷酸的M13mp2 DNA底物，与HeLa细胞胞质提取物孵育并随后用处理过的DNA转染细菌细胞后，观察到了广泛的修复。大多数（但不是全部）错配以及两个移码异源双链体都能被有效修复。对HeLa提取物和大肠杆菌中的修复进行的平行测量表明，这两个系统的修复特异性相似。分子中存在切口是HeLa细胞提取物中有效修复所必需的，含有切口的链是主要被修复的链。当比较提取物与异源双链体和同源双链体分子反应产生的放射性标记限制片段时，观察到了错配依赖性DNA合成。对于异源双链体底物，检测到了代表大约1000个碱基对区域且包含切口和错配的片段的特异性标记，而同源双链体则未检测到。修复反应在20分钟后完成，需要添加Mg2+、ATP和一个ATP再生系统，但不需要dNTPs，因为提取物中dNTPs的水平足够。DNA聚合酶β的抑制剂双脱氧胸苷5'-三磷酸不抑制错配特异性DNA合成。阿非迪霉素是DNA聚合酶α、δ和ε的抑制剂，它抑制提取物中的半保留复制和修复合成。丁基苯基-dGTP也抑制复制和修复合成，但浓度已知优先抑制DNA聚合酶α而不是δ或ε。这表明DNA聚合酶α可能在错配修复中起作用。
