Structure-activity relationships and kinetic analyses of polyribonucleotide inhibition of human cellular deoxynucleotide-polymerizing enzymes

Abstract

Fifteen polyribonucleotides were tested as inhibitors of terminal deoxynucleotidyltransferase (TDT) and DNA polymerase α, β, and γ activities. Seven were inhibitory and of these five contained purine residues and two contained pyrimidine residues. Poly(2′-O- methyladenylic acid) selectively inhibited TDT, poly(1,N 6- ethenoadenylic acid) selectively inhibited TDT and DNA polymerase γ, and the other inhibitors (poly(G), poly(I), poly(X), poly(U), and poly(4-thiouridylic acid)) were not selective except for poly(U) which did not inhibit TDT. Of the eight non-inhibitory polymers, three contained purine residues and five contained pyrimidine residues. The structure-activity relationships of these results indicate that polyribonucleotides containing purine residues generally are more inhibitory than those containing pyrimidine residues and that the type of substituent at the 6 position of the purine ring plays an important role in inhibition. In general, inhibitory polyribonucleotides were competitive inhibitors of TDT and DNA polymerase α and β activities with respect to template/initiator and non-competitive inhibitors with respect to substrate (deoxyribonucleoside triphosphates). In contrast, inhibition analysis of DNA polymerase γ activity with respect to template was not linear and thus complex. Inhibition of TDT and DNA polymerase α and β activities was linear with time and reversed by both simple dilution and extra template/initiator, but not reversed by extra enzyme except for inhibition by poly(4-thiouridylic acid). Inhibition of DNA polymerase γ activity was reversed by extra template (poly(A) · poly(dT) 12–18) but not by extra substrate (dTTP). Two inhibitions of DNA polymerase γ activity were progressive with time and reversed by extra enzyme (poly(G) and poly(I)); two other inhibitions were linear with time and not reversed by extra enzyme (poly(U) and poly(1,N 6- ethenoadenylic acid) ); and two additional inhibitions were linear with time and reversed by extra enzyme (poly(X) and poly(4-thiouridylic acid)). Preincubation of inhibitory polyribonucleotides with either TDT or DNA polymerases α or β or their respective template/initiators yielded the same results as non-preincubated reactions except for the preincubation of poly(4-thiouridylic acid) with DNA polymerase β which was more inhibitory than preincubation with template or non-preincubated reactions. Preincubation of inhibitory polyribonucleotides with DNA polymerase γ resulted in greater inhibition than preincubation with template or non-preincubated reactions. These results suggest that inhibitory polyribonucleotides generally are classical competitive inhibitors of TDT and DNA polymerase α and β activities with respect to template/initiator but that inhibition of DNA polymerase γ is more complex.