HERPES-SIMPLEX VIRUS TYPE-1 AND HUMAN DNA-POLYMERASE INTERACTIONS WITH 2'-DEOXYGUANOSINE 5'-TRIPHOSPHATE ANALOGS - KINETICS OF INCORPORATION INTO DNA AND INDUCTION OF INHIBITION

  • 15 November 1989
    • journal article
    • research article
    • Vol. 264  (32) , 19039-19044
Abstract
The ability of herpes simplex virus type 1 (HSV-1) DNA polymerase, HeLa polymerase .alpha., and HeLa polymerase .beta. to utilize several dGTP analogues has been investigated using a defined synthetic template primer. The relative efficiencies of the triphosphates of 9-[(2-hydroxyethoxy)methyl]guanine (acyclovir triphosphate, ACVTP), 9-[(1,3-dihydroxy-2-propoxy)methyl]guanine (ganciclovir triphosphate, DHPGTP), and 2'',3''-dideoxyguanosine (ddGTP) as substrates for the three polymerases were: HSV-1 polymerase, dGTP > ACVTP .simeq. DHPGTP > ddGTP; polymerase .alpha., dGTP > ACVTP .simeq. DHPGTP .mchgt. ddGTP; polymerase .beta., ddGTP > dGTP .mchgt. ACVTP .simeq. DHPGTP. The potent inhibition of HSV-1 polymerase by ACVTP has been shown previously to be due to the formation of a dead-end complex upon binding of the next 2''-deoxynucleoside 5''-triphosphate encoded by the template after incorporation of acyclovir monophosphate into the 3'' end of the primer (Reardon, J. E., and Spector, T. (1989) J. Biol. Chem. 264, 7405-7411). This mechanism was shown here to be a general mechanism for inhibition of polymerases by the obligate chain terminators, ACVTP and ddGTP. The ACVTP-induced inhibition was 30-fold more potent with HSV-1 polymerase than with polymerase .alpha.. This difference may contribute to the antiviral selectivity of this nucleotide analogue. The effect of ganciclovir monophosphate incorporation (a nonobligate chain terminator) on subsequent primer extension was also evaluated. With HSV-1 polymerase and polymerase .alpha., although there was a considerable reduction in the efficiency of utilization of the 3''-DHPGTP-terminal primer, contrasting kinetic behavior was observed. With HSV-1 polymerase, insertion of DHPGTP resulted in a significant reduction in Vmax for subsequent nucleotide incorporations. In contrast, with polymerase .alpha., a relatively small decrease in Vmax was accompanied by increased Km values for subsequent nucleotide incorporations.