Homogenous assays for Escherichia coli DnaB-stimulated DnaG primase and DnaB helicase and their use in screening for chemical inhibitors.
Escherichia coli DnaG primase is a single-stranded DNA-dependent RNA polymerase. Primase catalyzes the synthesis of a short RNA primer to initiate DNA replication at the origin and to initiate Okazaki fragment synthesis for synthesis of the lagging strand. Primase activity is greatly stimulated through its interaction with DnaB helicase. Here we report a 96-well homogeneous scintillation proximity assay (SPA) for the study of DnaB-stimulated E. coli primase activity and the identification of E. coli primase inhibitors. The assay uses an adaptation of the general priming reaction by employing DnaG primase, DnaB helicase, and ribonucleotidetriphosphates (incorporation of [(3)H]CTP) for in vitro primer synthesis on single-stranded oligonucleotide and M13mp18 DNA templates. The primase product is captured by polyvinyl toluene-polyethyleneimine-coated SPA beads and quantified by counting by beta-scintography. In the absence of helicase as a cofactor, primer synthesis is reduced by 85%. The primase assay was used for screening libraries of compounds previously identified as possessing antimicrobial activities. Primase inhibitory compounds were then classified as direct primase inhibitors or mixed primase/helicase inhibitors by further evaluation in a specific assay for DnaB helicase activity. By this approach, specific primase inhibitors could be identified.
Zhang
Yi
Y
Pharmaceutical Research Institute, Bristol-Myers Squibb Company, Wilmington, DE 19880, USA.
Yang
Fude
F
Kao
Yeh-Chih
YC
Kurilla
Michael G
MG
Pompliano
David L
DL
Dicker
Ira B
IB
D001426:Bacterial Proteins; D004265:DNA Helicases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D019915:DNA Primase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D053488:DnaB Helicases; D004735:Energy Transfer; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004926:Escherichia coli / Q000201:enzymology*; D005454:Fluorescence Polarization; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D009841:Oligonucleotides / Q000378:metabolism; D012588:Scintillation Counting; D013379:Substrate Specificity
D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D019384:Nucleic Acid Synthesis Inhibitors; D009841:Oligonucleotides; D019915:DNA Primase; D004265:DNA Helicases; D053488:DnaB Helicases