Alternate pathways of DNA replication in Escherichia coli.
We have described the pcbA1 mutation which enables E. coli cells to replicate DNA in the absence of a functional dnaE gene product if DNA polymerase I (the polA gene product) is present. The pcbA1 mutation phenotypically suppresses multiple dnaEts and dnaEam alleles. The pcbA1/PolI replication pathway differs from normal in sensitivity to certain DNA-damaging agents such as methylmethane sulfonate (MMS) and a lack of damage-directed mutagenesis. We report here cloning of the pcbA1 gene in a multicopy plasmid. The pcbA1 mutation is detected only in cis; therefore, cloning necessitated gene eviction. The pcbA1 gene lies closely- linked to gyrB. We have demonstrated the physical presence of DNA polymerase I in the replicating holoenzyme complex by immunoblotting using dnaEam strains. We conclude that E. coli has two alternate replisome structures: REP-A, in which DNA polymerase I is the functional synthetic subunit; and REP-E, in which the alpha-subunit, product of the dnaE gene, is functional. To investigate further the role of individual DNA polymerases in replication, we have isolated the polB gene on multicopy plasmids.
Bryan
S
S
Department of Cell Biology, Baylor College of Medicine, Houston, TX 77030.
Chen
H
H
Sun
Y
Y
Moses
R E
RE
D000483:Alleles; D001426:Bacterial Proteins / Q000235:genetics; D010582:Bacteriophage lambda / Q000235:genetics; D003001:Cloning, Molecular; D004256:DNA Polymerase I / Q000378:metabolism; D004257:DNA Polymerase II / Q000378:metabolism; D004258:DNA Polymerase III / Q000378:metabolism; D004261:DNA Replication; D004269:DNA, Bacterial / Q000096:biosynthesis*; D004926:Escherichia coli / Q000235:genetics*; D005798:Genes, Bacterial; D015151:Immunoblotting; D009154:Mutation; D010957:Plasmids
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004256:DNA Polymerase I; D004257:DNA Polymerase II; D004258:DNA Polymerase III