Molecular Pharmacology, Vol 8, 362-370, Copyright © 1972 by the American Society for Pharmacology and Experimental Therapeutics

Kinetic and Template Studies with 1--D-Arabinofuranosylcytosine 5'-Triphosphate and Mammalian Deoxyribonucleic Acid Polymerase

¹ McGill University Cancer Research Unit, Montreal, Quebec, Canada

Kinetic and template studies have been performed with 1--D-arabinofuranosylcytosine 5'-triphosphate (araCTP), dCTP, and calf thymus DNA polymerase. Using denatured DNA as the template, the estimated apparent K_m values for araCTP and dCTP were about the same; however, the estimated apparent V_max value for dCTP was about 8 times greater than the apparent V_max for araCTP. Both araCTP and dCTP appeared to complete for the same catalytic site of DNA polymerase. When poly(dA-dT) was used as the template, araCTP did not produce any detectable inhibition of the incorporation of [³H]dTTP into acid-insoluble material. DNA Polymerase catalyzed the incorporation of [³H]araCTP into poly dC:poly dG, but not into poly(dA-dT). DNA containing [-³²P]arabinofuranosylcytosine 5'-monophosphate ([-³²P]araCMP) was synthesized enzymatically, using denatured DNA and DNA polymerase. Following the digestion of this [³²P]DNA with micrococcal nuclease and spleen phosphodiesterase to deoxynucleoside 3'-monophosphates, the radioactivity from [-³²P]araCMP appeared in 3'-dAMP, 3'-dGMP, 3'-dTMP, and 3'-dCMP, suggesting that DNA polymerase could catalyze the formation of a phosphodiester bond between araCMP and each of the deoxynucleotides present in the DNA template.

The data from studies on the rate of release of [-³²P]araCMP and [³H]dCMP from DNA by snake venom phosphodiesterase and the incorporation of araCTP and dCTP into denatured, sonicated DNA are consistent with the proposal that the incorporation of araCTP into DNA in the reaction catalyzed by DNA polymerase produces termination of polynucleotide chain growth.

Note:
ACKNOWLEDGMENT I would like to thank Miss Aurora Labitan for her excellent technical assistance in this work.