Aphidicolin inhibits DNA polymerizing activity but not nucleolytic activity of Escherichia coli DNA polymerase II.
We have purified the DNA polymerase II of Escherichia coli from the recombinant strain carrying the plasmid which encodes the polB gene. We confirmed that the purified protein, of molecular weight 90,000, possesses a 3'----5' exonuclease activity in addition to DNA polymerizing activity in a single polypeptide. Its DNA polymerizing activity was sensitive to the drug aphidicoline, which is a specific and direct inhibitor of the alpha-like DNA polymerases including eukaryotic replicative DNA polymerases. Aphidicolin had no detectable effect on the 3'----5' exonuclease activity. The inhibition by aphidicolin on the polymerizing activity of polymerase II was competitive with respect to dNTP and uncompetitive with respect to template DNA. This mode of action is the same as that on eukaryotic DNA polymerase alpha. The apparent Ki value calculated from Lineweaver-Burk plots was 55.6 microM.
Ishino
Y
Y
Biotechnology Research Laboratories, Shiga, Japan.
Iwasaki
H
H
Fukui
H
H
Mineno
J
J
Kato
I
I
Shinagawa
H
H
D016590:Aphidicolin / Q000494:pharmacology*; D004247:DNA / Q000378:metabolism; D004257:DNA Polymerase II / Q000037:antagonists & inhibitors* / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000378:metabolism; D007700:Kinetics; D009711:Nucleotides / Q000378:metabolism; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors
D009711:Nucleotides; D011994:Recombinant Proteins; D016590:Aphidicolin; D004247:DNA; D004257:DNA Polymerase II; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V