Mutagenesis and Plasmids

A rapid method for cloning mutagenic DNA repair genes: isolation of umu-complementing genes from multidrug resistance plasmids R391, R446b, and R471a

“…First, research by others had suggested that they might contain umu analogs (5,39,54); second, unlike the IncN plasmid R46, which encodes mucAB; the IncI plasmid TP110, which encodes imppCAB, and the cryptic pSLT plasmid encoding samAB (all of which were isolated from Salmonella strains [23,30,38]), R391, R446b, and R471a were originally isolated from Providencia rettgeri (10, 36a), Morganella morganii (16, 36a), and Serratia marcescens (17), respectively, and therefore might have evolved differently from the previously identified Salmonella plasmids; and third, they appeared to promote mutagenesis functions to various degrees. In general, previous reports have suggested that R446b is more efficient at promoting mutagenesis functions than R471a (yet both come from the same plasmid incompatibility group) and both are much better than R391 (5,28,54). We hypothesized that if we were successful in cloning the umu-like genes from these plasmids, our results might provide us with a better insight into the structure-function relationship between these and the previously identified mutagenesis proteins.…”

Characterization of the umu-complementing operon from R391

“…Finally, it should be noted that the high level of mutagenesis promoted by the cloned rumAB (R391) operon is in stark contrast to the limited ability of the native R391 plasmid to promote mutagenesis functions (8,27,28,39,53). One obvious explanation for this difference is that the UV sensitization gene(s) carried by R391 (and other IncJ plasmids) (35,39,53) masks the inherent ability of rumAB (R391) to exhibit mutagenic DNA repair.…”

“…These so-called mutagenesis proteins can be expressed chromosomally, like the UmuDC proteins from Escherichia coli and Salmonella typhimurium (19,37,48,52), or episomally from certain naturally occurring plasmids (23,31,37,38). Indeed, R plasmids from over 10 different incompatibility groups can restore or increase the mutation frequency in a variety of E. coli strains, suggesting that they carry functionally active Umu homologs (8,27,28,39,53).…”

The enhanced mutagenic potential of the MucAB proteins correlates with the highly efficient processing of the MucA protein

“…Genetic experiments suggest that the UmuDC proteins may promote DNA chain elongation after a misincorporation event opposite DNA lesions (8,9). The occurrence of these mutagenic DNA repair genes is widespread; some are encoded by chromosomal genes like those of E. coli and Salmonella typhimurium (2, 18, 19, 37, 45, 50-52, 56), while others are found on extrachromosomal multidrug resistance plasmids (R plasmids) (24,32,33,36,53). Of these R plasmids, pKM101, a smaller variant of plasmid R-46, appears to be the most efficient at promoting cellular mutagenesis (32,44).…”

“…The occurrence of these mutagenic DNA repair genes is widespread; some are encoded by chromosomal genes like those of E. coli and Salmonella typhimurium (2, 18, 19, 37, 45, 50-52, 56), while others are found on extrachromosomal multidrug resistance plasmids (R plasmids) (24,32,33,36,53). Of these R plasmids, pKM101, a smaller variant of plasmid R-46, appears to be the most efficient at promoting cellular mutagenesis (32,44). The enhanced mutability seen in certain S. typhimurium strains carrying pKM101 after exposure to a variety of DNA-damaging agents has led to its incorporation in the Ames tester strains used to detect environmental mutagens (28).…”