Isolation of a DNA Polymerase α-associated Regulatory Protein from Calf Thymus

Abstract A regulatory protein for DNA polymerase α, responsive to noncomplementary deoxyribonucleoside triphosphates, has been isolated from calf thymus. The regulatory protein was separated from DNA polymerase α using Affi-Gel Blue and gel filtration. The regulatory protein had a molecular weight of approximately 70,000 as determined by gel filtration, and its activity was nondialyzable, heat labile, and abolished by pronase treatment. In the presence of regulatory protein, DNA polymerase α activity, measured by using polydeoxyadenylate-oligodeoxythymidylate as template primer, was inhibited by 2′-deoxyguanosine 5′-triphosphate in a parabolic-competitive fashion [K i = 15 ± 1 (S.E.) µm] and by 2′-deoxycytidine 5′-triphosphate in a linear-competitive manner (K i = 162 ± 23 µm). Neither the four natural ribonucleoside triphosphates nor 2′-deoxyadenosine 5′-triphosphate inhibited the DNA polymerase-regulatory protein system to any significant extent. The regulatory protein by itself had no effect on either DNA polymerase α activity or the K m for template primer. These results indicate that deoxyribonucleoside triphosphate pools may be involved in the regulation of cellular DNA synthesis through a direct effect on DNA polymerization.