BIOCHEMISTRY OF TERMINAL DEOXYNUCLEOTIDYLTRANSFERASE - MECHANISM OF MANGANESE-DEPENDENT INHIBITION BY DEOXYADENOSINE 5'-TRIPHOSPHATE AND BIOLOGICAL IMPLICATIONS

We have shown that terminal deoxynucleotidyltransferase (TdT) catalyzed DNA synthesis is strongly inhibited in the presence of all the four ribonucleoside triphosphates, ATP being the most potent [Modak, M. J. (1978) Biochemistry 77, 3116]. In an activated DNA primed reaction, the effective order of triphosphate inhibition is A>G>C>U. Furthermore, inhibition appears to be more severe in the presence of Mn2+ when compared to that observed in the presence of Mg2+. Extension of these findings to de-oxynucleoside triphosphates revealed that only dATP, in the presence of Mn2+, strongly inhibits TdT-catalyzed incorporation of the remaining three triphosphates. In contrast to the inhibitory effect of rNTPs and Mn-dATP on TdT, replicative DNA polymerases are resistant to the addition of rNTPs and are either unaffected or stimulated by the addition of dNTPs. An investigation into the mechanism of Mn2+-dependent dATP inhibition of TdT revealed that the observed inhibition is a result of multiple effects involving the three important components of the catalysis, namely, enzyme, primer and substrate. The rates of incorporation of Mn-dATP are ~ 50-fold lower than those observed with Mn-dGTP, although the affinity for binding Mn-dATP to enzyme is approximately 1-2 orders of magnitude higher than that of Mn-dGTP. This probably results in the blockage of the substrate binding site on TdT by Mn-dATP. Increasing the primer concentration affords partial protection from dATP inhibition; kinetics studies performed with changing primer concentration and inhibitor dATP have yielded noncompetitive plots. A direct involvement of Mn-dATP as a competitor for the substrate dGTP for binding to the enzyme is revealed by the partial competitive modes of inhibition observed with kinetics studies performed by using these two substrates. Furthermore, the addition of Mn2+ to a reaction proceeding in the presence of Mg2+ may be instantly inhibited. Biological implications of adenine nucleotide mediated inhibition of TdT have been discussed in light of the present results and the possible involvement of TdT in the differentiation of lymphocytes and immunodeficiency. © 1979, American Chemical Society. All rights reserved.