Inhibitory effects of various 2',3'-dideoxynucleoside 5'-triphosphates on the utilization of 2'-deoxynucleoside 5'-triphosphates by terminal deoxynucleotidyltransferase from calf thymus.
Inhibitory effects of four 2',3'-dideoxynucleoside 5'-triphosphates (i.e., those of 2',3'-dideoxyadenosine, 2',3'-dideoxycytidine, 2',3'-dideoxyguanosine and 2',3'-dideoxythymidine, ddATP, ddCTP, ddGTP and ddTTP, respectively) on the utilization of 2'-deoxynucleoside 5'-triphosphates (dNTPs) by terminal deoxynucleotidyltransferase (TdT) from calf thymus were examined in comparison with those of ribonucleoside triphosphates. All four dideoxy compounds strongly inhibited the dNTP utilization by TdT. The inhibitions by these dideoxy compounds were stronger with (dA) than with heat denatured DNA as the primer. Inhibition of TdT by any of these compounds was due to competition with the natural substrate dNTP for the same substrate binding site of the enzyme. Ki values for these dideoxynucleoside triphosphates (ddNTPs) were much lower than the Km values for the corresponding dNTPs indicating a higher affinity of TdT for ddNTP than for dNTP. Among the Ki values determined with (dA) as the primer, those for ddATP (1.3 microM), ddGTP (1.3 microM) and ddCTP (2.8 microM) were lower than that for ddTTP (17.5 microM). With heat-denatured DNA, the Ki for ddATP (2.9 microM) was smaller than those for the other dideoxynucleotides (7.3-35 microM). Also, both the Km for dATP and the Ki for ATP were the lowest among those of the respective deoxynucleotide substrates and ribonucleotide inhibitors. These results indicate that TdT has a stronger affinity for the adenine base of all of the ribo-, deoxyribo-, and dideoxyribo-nucleoside triphosphates than for the other bases of these nucleotides.
Ono
K
K
Laboratory of Viral Oncology, Aichi Cancer Center Research Institute, Nagoya, Japan.
D000818:Animals; D002417:Cattle; D054306:Dideoxynucleotides; D007700:Kinetics; D009711:Nucleotides / Q000378:metabolism / Q000494:pharmacology*; D009713:Nucleotidyltransferases; D013379:Substrate Specificity; D013942:Thymine Nucleotides / Q000378:metabolism*; D013950:Thymus Gland / Q000187:drug effects / Q000201:enzymology*
D054306:Dideoxynucleotides; D009711:Nucleotides; D013942:Thymine Nucleotides; D009713:Nucleotidyltransferases; C017216:2',3'-dideoxythymidine triphosphate