PolC-type polymerase III of Streptococcus pyogenes and its use in screening for chemical inhibitors.
The polC gene from Streptococcus pyogenes (S. pyogenes, strain SF370) has been cloned and expressed in Escherichia coli (E. coli) as a fusion protein containing an N-terminal histidine tag. The purified recombinant enzyme showed an apparent molecular mass of 160 kDa on SDS-PAGE and a specific activity of 3.5 nmol/min/mg when assayed in the presence of calf thymus DNA and the four deoxyribonucleoside triphosphates. This activity was inhibited by TMAU, a specific inhibitor of PolC. To facilitate kinetic studies, and high-throughput assays, a double-stranded oligo DNA primer/template was used as a substrate. The minimum requirement for the length of the substrate was a 20-base oligo primer annealed to a 35-base template. PolC activity was detected either by a filter-binding format or by a novel homogeneous scintillation proximity assay (SPA). Sensitivity to inhibition by anilinouracil analogs was improved by incorporating three deoxycytidines in the template strand as the first 3 bases to be copied by the polymerase. Inhibition of PolC activity by trimethyleneanilinouracil by the filtration and SPA methods gave comparable results, but the SPA assay uses less radioactive label, is less time-consuming, and is amenable to high-throughput formatting.
Yang
Fude
F
Bristol-Myers Squibb Company, Pharmaceutical Research Institute, Wilmington, Delaware 19880, USA.
Dicker
Ira B
IB
Kurilla
Michael G
MG
Pompliano
David L
DL
D000818:Animals; D001426:Bacterial Proteins; D001483:Base Sequence; D002417:Cattle; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004258:DNA Polymerase III / Q000032:analysis* / Q000037:antagonists & inhibitors*; D004269:DNA, Bacterial / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000032:analysis*; D004353:Drug Evaluation, Preclinical; D004791:Enzyme Inhibitors / Q000302:isolation & purification / Q000494:pharmacology; D005798:Genes, Bacterial; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D019384:Nucleic Acid Synthesis Inhibitors; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000037:antagonists & inhibitors; D012588:Scintillation Counting; D013297:Streptococcus pyogenes / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity; D014498:Uracil / Q000031:analogs & derivatives / Q000494:pharmacology
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004791:Enzyme Inhibitors; D019384:Nucleic Acid Synthesis Inhibitors; D011993:Recombinant Fusion Proteins; D014498:Uracil; D004247:DNA; C446929:PolC protein, bacteria; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase