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Isolation of a DNA Polymerase -associated Regulatory Protein from Calf Thymus¹

Department of Experimental Therapeutics, Grace Cancer Drug Center, Roswell Park Memorial Institute, Buffalo, New York 14263

A regulatory protein for DNA polymerase , responsive to noncomplementary deoxyribonucleoside triphosphates, has been isolated from calf thymus. The regulatory protein was separated from DNA polymerase 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 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 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.

¹ Supported in part by USPHS Grant Ca-17156 from the National Cancer Institute and the B. B. Hoffman Memorial Trust.

² This work is submitted to the Program of Pharmacology, Roswell Park Memorial Institute Division, State University of New York at Buffalo, in partial fulfillment of the requirements for the Ph.D. degree.

³ To whom requests for reprints should be addressed, at Department of Experimental Therapeutics, Grace Cancer Drug Center, Roswell Park Memorial Institute, 666 Elm Street, Buffalo, N. Y. 14263.

Received 1/31/79. Accepted 7/25/79.