Bacillus subtilis DNA polymerase III: complete sequence, overexpression, and characterization of the polC gene.
Genomic DNA encompassing polC, the structural gene specifying Bacillus subtilis DNA polymerase III (PolIII), was sequenced and found to contain a 4311-bp open reading frame (ORF) encoding a 162.4-kDa polypeptide of 1437 amino acids (aa). The ORF was engineered into an Escherichia coli expression plasmid under the control of the coliphage lambda repressor. Derepression of E. coli transformants carrying the recombinant vector resulted in the high-level synthesis of a recombinant DNA polymerase indistinguishable from native PolIII. N-terminal aa sequence analysis of the recombinant polymerase unequivocally identified the 4311-bp ORF as that of polC. Comparative aa sequence analysis indicated significant homology of the B. subtilis enzyme with the catalytic alpha subunit of the E. coli PolIII and, with the exception of an exonuclease domain, little homology with other DNA polymerases. The respective sequences of the mutant polC alleles, dnaF and ts-6, were identified, and the expression of specifically truncated forms of polC was exploited to assess the dependence of polymerase activity on the structure of the enzyme's C terminus.
Hammond
R A
RA
Department of Pharmacology, University of Massachusetts Medical School, Worcester 01655.
Barnes
M H
MH
Mack
S L
SL
Mitchener
J A
JA
Brown
N C
NC
D000483:Alleles; D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D003062:Codon; D004258:DNA Polymerase III / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004269:DNA, Bacterial; D004259:DNA-Directed DNA Polymerase / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010957:Plasmids; D014176:Protein Biosynthesis; D012689:Sequence Homology, Nucleic Acid; D014169:Transformation, Bacterial
D003062:Codon; D004269:DNA, Bacterial; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase