Cloning of mutator genes and identification of their products

To elucidate molecular mechanisms leading to the increment of mutation frequency by mutator genes, we have cloned the dnaQ and uvrD genes of Escherichia coli and identified their gene products. By in vitro recombination hybrid plasmids capable of complementing a dnaQ mutation were constructed. The dnaQ+ plasmids consist of a full-length pBR322 DNA and a 1.5-kilobase DNA fragment derived from the E. coli chromosome. Specific labeling of plasmid-encoded proteins by the maxicell method revealed that the 1.5-kilobase insert codes for two proteins, one with a molecular weight of 25,000 and the other with a weight of 21,000. Because insertion of the gamma delta sequence into the dnaQ gene of the plasmid resulted in disappearance of the 25,000-dalton protein, it was concluded that that protein is the dnaQ gene product. The 21,000-dalton protein was identified as RNase H. The uvrD (uvrE, recL, mutU, and pdeB) gene has been cloned with phage lambda as vector. The increased sensitivity to ultraviolet light, high mutability and conditional lethality of uvrD- strains and their derivatives were all suppressed by lysogenization of the mutant cells with lambda uvrD+. In addition to the uvrD gene, lambda uvrD+ carried the corA gene that controls transport of Mg2+, Mn2+, and Co2+ through the cell membrane. By analyzing proteins produced by the transducing phages, the uvrD and corA gene products were identified as a 75,000-dalton protein and a 37,000-dalton protein, respectively.