Mechanisms of mutagenesis in the Escherichia coli mutator mutD5: role of DNA mismatch repair.

To investigate the mechanisms of spontaneous mutation in the Escherichia coli mutD5 mutator strain, 502 mutations generated in this strain in the N-terminal part of the lacI gene were sequenced (i-d mutations). Since the mutator strength of this strain depends on the medium in which it grows, mutations were analyzed in both minimal medium (moderate mutator activity) and rich medium (high mutator activity). In either case, 95% of all mutations were base substitutions and 5% were single-base deletions. However, the nature and site distribution of the base substitutions differed dramatically for the two conditions. In minimal medium (mutation frequency, 480-fold above background), a majority (62%) were transversions, notably A.T----T.A at three 5'-GTGG-3' sequences. Most (64%) of the transitions under this condition occurred at specific sequences that are suggestive of a "dislocation" type of mutagenesis. In rich medium (mutation frequency, 37,000-fold above background), 90% of the base substitutions were transitions. These observations suggest that different modes of mutagenesis operate under the two conditions. mutD5 cells have been reported to be defective in exonucleolytic proofreading during DNA replication. The present data suggest that mutD cells in rich medium also suffer a defect in mutHLS-encoded mismatch correction. This hypothesis was confirmed by the direct measurement of mismatch repair in mutD5 cells by transfection of M13mp2 heteroduplex DNA.