Abstract
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.
Highlights
Has been detected in HeLa cell extractsU. sing a variety 8)
The assay (41) is based on the analysis of plaque color phenotypes resulting from the transfection of a mutS E. coli strain (NR9162, which is defective in methyl-directed mismatch repair)with purified M13mp2 heteroduplex DNAs that have been treated with human cell extracts
We have presented the initial characterization of a general mismatch repair system in an extract of human HeLa cells which has been shown previously to be replication competent (32)
Summary
A similar study demonstrated heterogeneityof mismatch repair in simian cells fora wide variety of mispairs, suggestingthat a general mismatch repair pathway may exist (23).A specific repair pathway was discovered in simian cells whereby G’T mispairswere preferentiallyrepaired to G C (24,25),a system analogous to the very short patch repair pathway in E. coli. This system has been observed in human cell extracts (26). Which is capable of strand discriminationby utilizing a strand break to direct repair of the strand containing the nick
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