Thermally denaturing high-performance liquid chromatography analysis of primase activity.
Prokaryotic primase, a DNA-dependent RNA polymerase, is a target of interest for the development of novel antibiotics. A new assay was developed to evaluate the inhibition of primase activity while avoiding the limitations of existing assays that require the incorporation of radiolabeled nucleotides into the growing primer followed by electrophoretic separation and autoradiography or scintillation counting. These existing technologies are either time consuming or unable to give detailed information on the kinetics, size, and nature of the primers synthesized. To address these issues in a nonradioactive manner, a thermally denaturing high-performance liquid chromatography (HPLC) assay was developed that was able to (1) measure the two modes of primase activity (de novo and overlong primer synthesis), (2) quantitate de novo primer synthesis kinetics yielding a rate constant of 0.00251 s(-1), and (3) determine that dNTPs inhibited primase activity with an IC50 of 9.5 microM. In addition, the differential elution properties of short DNA and RNA oligonucleotides on an alkylated nonporous polystyrene-divinylbenzene copolymer microsphere bead column were determined. The thermally denaturing HPLC assay provides rapid quantitative analysis of primase function and qualitative analysis of activity with regard to the nature of the primers synthesized.
Koepsell
Scott
S
Department of Microbiology and Pathology, University of Nebraska Medical Center, Omaha, NE 68101, USA.
Bastola
Dhundy
D
Hinrichs
Steven H
SH
Griep
Mark A
MA
D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D019915:DNA Primase / Q000378:metabolism*; D004261:DNA Replication; D004926:Escherichia coli / Q000201:enzymology*; D006358:Hot Temperature; D009696:Nucleic Acids / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D011489:Protein Denaturation; D012680:Sensitivity and Specificity; D013698:Templates, Genetic
D009696:Nucleic Acids; D019915:DNA Primase