Studies on the purification of DNA polymerase α and the control mechanism of enzyme activity in NIH3T3 cells

Abstract

It has been known that DNA polymerase a plays $\alpha$ major role in DNA replication of eukaryotes, moreover has function of repairing damaged DNA. To purify DNA polymerase $\alpha$, two different protocols were used;First was conventional chromatographic method that has been generally used for protein purification, Second was immunoaffinity chromatography using monoclonal antibody which specifically binds to human DNA polymerase $\alpha$. Of purified DNA polymerase $\alpha$ subunits, $>$190kDa protein was degraded by protease. Its activity was reduced by aphidicolin, specific DNA polymerase $\alpha$ inhibitor, but not by dideoxythymidine triphosphate which is an inhibitor of other DNA polymerases. DNA polymerase a activity in cell was changed during the progression of cell cycle. Enzyme activity was increased slightly before DNA replication(S phase). When NIH3T3 cells were irradiated by UV light to damage chromosomal DNA, DNA polymerase $\alpha$ activity was increased in nuclear fraction. When cell were treated with PMA(Phorbol myristate acetate), protein kinase C activator and modulator of cell division and differentiation, DNA polymerase $\alpha$ activity increased in cytoplasmic and nuclear fractions. DNA polymerase $\alpha$ is involved in DNA repair and DNA replicaiton, and the increment of enzyme activity is a result of posttrannnslational modification. This implicates that DNA polymerase $\alpha$ activity might be controlled by protein kinase C in vivo.