Directionality of DNA replication fork movement strongly affects the generation of spontaneous mutations in Escherichia coli.
Using a pair of plasmids carrying the rpsL target sequence in different orientations to the replication origin, we analyzed a large number of forward mutations generated in wild-type and mismatch-repair deficient (MMR(-)) Escherichia coli cells to assess the effects of directionality of replication-fork movement on spontaneous mutagenesis and the generation of replication error. All classes of the mutations found in wild-type cells but not MMR(-) cells were strongly affected by the directionality of replication fork movement. It also appeared that the directionality of replication-fork movement governs the directionality of sequence substitution mutagenesis, which occurred in wild-type cells at a frequency comparable to base substitutions and single-base frameshift mutations. A very strong orientation-dependent hot-spot site for single-base frameshift mutations was discovered and demonstrated to be caused by the same process involved in sequence substitution mutagenesis. It is surprising that dnaE173, a potent mutator mutation specific for sequence substitution as well as single-base frameshift, did not enhance the frequency of the hot-spot frameshift mutation. Furthermore, the frequency of the hot-spot frameshift mutation was unchanged in the MMR(-) strain, whereas the mutHLS-dependent mismatch repair system efficiently suppressed the generation of single-base frameshift mutations. These results suggested that the hot-spot frameshift mutagenesis might be initiated at a particular location containing a DNA lesion, and thereby produce a premutagenic replication intermediate resistant to MMR. Significant numbers of spontaneous single-base frameshift mutations are probably caused by similar mechanisms.
Yoshiyama
K
K
Department of Molecular Biology, Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara, 630-0101, Japan.
Higuchi
K
K
Matsumura
H
H
Maki
H
H
D000251:Adenosine Triphosphatases; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D020137:Base Pair Mismatch / Q000235:genetics; D001483:Base Sequence; D004252:DNA Mutational Analysis; D004258:DNA Polymerase III / Q000235:genetics / Q000378:metabolism; D004261:DNA Replication / Q000235:genetics*; D004268:DNA-Binding Proteins; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D029968:Escherichia coli Proteins; D016368:Frameshift Mutation / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics; D008957:Models, Genetic; D008969:Molecular Sequence Data; D051719:MutS DNA Mismatch-Binding Protein; D016296:Mutagenesis / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D017354:Point Mutation / Q000235:genetics; D016133:Polymerase Chain Reaction; D000091243:Ribosomal Protein S9
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D000091243:Ribosomal Protein S9; C000717190:RpsI protein, E coli; C074904:DNA polymerase III, alpha subunit; D004258:DNA Polymerase III; D000251:Adenosine Triphosphatases; D051719:MutS DNA Mismatch-Binding Protein; C497175:MutS protein, E coli