Purification of CV-1 polymerase delta and studies implicating both polymerase delta and alpha in DNA replication

DNA polymerase delta has been identified and purified from VC-1 cells. This is the first system reported where polymerase delta has been purified and where its role in DNA synthesis can be characterized. DNA polymerase inhibitors, including aphidicolin, butylphenyldeoxyguanosine triphosphate (BuPdGTP), and a monoclonal antibody which specifically inhibits DNA polymerase alpha activity were used to investigate the role of polymerases delta and alpha in DNA replication in lysolecithin permeabilized CV-1 cells. The effects of these inhibitors on purified polymerases delta and alpha were also determined. Aphidicolin was a potent inhibitor of DNA replication and DNA polymerase delta and alpha. However, concentrations of BuPdGTP which totally abolished DNA polymerase alpha activity were much less effective in reducing DNA replication and the activity of DNA polymerase delta. A monoclonal antibody specific for polymerase alpha reduced DNA replication in permeable cells 60-70%. The remaining replication activity appeared to be attributed to polymerase delta, because it was aphidicolin sensitive and resistant to low concentrations of BuPdGTP. This information, and the kinetics of inhibition of replication displayed by these inhibitors, indicate that in addition to polymerase alpha, polymerase delta also plays a significant role in mammalian DNA replication.