DnaG primase plays an essential role in lagging strand DNA synthesis by its ability to synthesize short RNA polymers. Our lab has developed the simple primer synthesis assay to measure the activity of this enzyme. This assay is termed simple in that the only requirements are: a short ssDNA template containing the preferred initiating sequence d(CTG), ribonucleotides, Mg 2+ and primase. Using a radiolabeled nucleotide. one can quantitate the RNA polymer products after electrophoresis on a DNA sequencing gel and exposure to a Phosphoimager plate made by Molecular Dynamics. Radioactive standards are simultaneously exposed to the plate to allow for a direct relationship between pixel intensity of primer bands and actual fmol UTP incorporation. Use of this assay has shown DnaG primase to be the slowest polymerase known. Because of this, our lab has set out to find a possible stimulant of primase. Singlestranded DNA binding protein was found to inhibit rather then stimulate, indicating that it binds the ssDNA template in such a way that primase no longer has access to the initiating trinucleotide. DnaB helicase, however, was found to cause nearly a 100-fold increase in primer production. This means that DnaB helicase is able to stimulate primase activity on ssDNA that does not have any secondary structure.
|Original language||English (US)|
|State||Published - 1997|
ASJC Scopus subject areas
- Molecular Biology