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Action of Partially Thiolated Polynucleotides on the DNA Polymerase from Regenerating Rat Liver¹

Department of Biochemical Pharmacology, State University of New York at Buffalo, Buffalo, New York 14214

The effects of partially thiolated polynucleotides on the DNA polymerase from regenerating rat liver were investigated. The enzyme was isolated from the nuclear fraction essentially according to the method of Baril et al.; it was characterized as the polymerase on the basis of its response to synthetic templates and its inhibition with N-ethylmaleimide. Although polycytidylic acid had no effect on the DNA polymerase either as a template or as an inhibitor, partially thiolated polycytidylic acid (MPC) was found to be a potent inhibitor, its activity being directly related to its extent of thiolation (percentage of 5-mercaptocytidylate units in the polymer). In comparison, the DNA polymerase ß which was purified from normal rat liver nuclear fraction, was much less sensitive to inhibition by MPC. Analysis of the inhibition of the polymerase by the method of Lineweaver and Burk showed that the inhibitory action of MPC was competitively reversible with the DNA template, but the binding of the 7.2%-thiolated MPC to the enzyme was much stronger than that of the template (K_i/K_m < 0.03). Polyuridylic acid as such showed some inhibitory activity which increased on partial thiolation, but the 8.4%-thiolated polyuridylic acid was less active than the 7.2% MPC. When MPC was annealed with polyinosinic acid, it lost 80% of its inhibitory activity in the double-stranded configuration. However, 1 to 2%-thiolated DNA isolates were significantly more potent inhibitors than were comparable (1.2%-thiolated) MPC and showed competitive reversibility with the unmodified (but "activated") DNA template. These results indicate that the inhibitory activities of partially thiolated polynucleotides depend not only on the percentage of 5-mercapto groups but also on the configuration, base composition, and other specific structural properties.

¹ This work was supported by American Cancer Society Research Grant CH-20C and NIH Grant CA-06695-13 from the National Cancer Institute, USPHS.

² National Cancer Institute Research Fellow (Award IF 22 CA-02065).

Received 6/ 1/76. Accepted 9/ 1/76.

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