Escherichia coli DNA polymerase IV mutator activity: genetic requirements and mutational specificity.
The dinB gene of Escherichia coli is known to be involved in the untargeted mutagenesis of lambda phage. Recently, we have demonstrated that this damage-inducible and SOS-controlled gene encodes a novel DNA polymerase, DNA Pol IV, which is able to dramatically increase the untargeted mutagenesis of F' plasmid. At the amino acid level, DNA Pol IV shares sequence homologies with E. coli UmuC (DNA Pol V), Rev1p, and Rad30p (DNA polymerase eta) of Saccharomyces cerevisiae and human Rad30A (XPV) proteins, all of which are involved in translesion DNA synthesis. To better characterize the Pol IV-dependent untargeted mutagenesis, i.e., the DNA Pol IV mutator activity, we analyzed the genetic requirements of this activity and determined the forward mutation spectrum generated by this protein within the cII gene of lambda phage. The results indicated that the DNA Pol IV mutator activity is independent of polA, polB, recA, umuDC, uvrA, and mutS functions. The analysis of more than 300 independent mutations obtained in the wild-type or mutS background revealed that the mutator activity clearly promotes single-nucleotide substitutions as well as one-base deletions in the ratio of about 1:2. The base changes were strikingly biased for substitutions toward G:C base pairs, and about 70% of them occurred in 5'-GX-3' sequences, where X represents the base (T, A, or C) that is mutated to G. These results are discussed with respect to the recently described biochemical characteristics of DNA Pol IV.
Wagner
J
J
Division of Genetics and Mutagenesis, National Institute of Health Sciences, Setagaya-ku, Tokyo 158-8501, Japan.
Nohmi
T
T
D000251:Adenosine Triphosphatases; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D010582:Bacteriophage lambda / Q000235:genetics*; D020137:Base Pair Mismatch; D001483:Base Sequence; D019951:DNA Polymerase beta / Q000378:metabolism*; D004268:DNA-Binding Proteins; D004352:Drug Resistance, Microbial; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics* / Q000821:virology; D029968:Escherichia coli Proteins; D016368:Frameshift Mutation; D005838:Genotype; D006801:Humans; D008969:Molecular Sequence Data; D051719:MutS DNA Mismatch-Binding Protein; D016296:Mutagenesis; D010957:Plasmids; D017354:Point Mutation; D012293:Rifampin / Q000494:pharmacology; D013014:SOS Response, Genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C094399:DinB protein, E coli; D029968:Escherichia coli Proteins; D019951:DNA Polymerase beta; D000251:Adenosine Triphosphatases; D051719:MutS DNA Mismatch-Binding Protein; C497175:MutS protein, E coli; D012293:Rifampin