9-[2-(phosphonomethoxy)ethyl]adenine diphosphate (PMEApp) as a substrate toward replicative DNA polymerases α, δ, ε, and ε*

The diphosphoryl derivative of the acyclic nucleotide phosphonate analog 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA), found previously to weakly inhibit DNA pol delta/proliferating cell nuclear antigen, was studied as a substrate for pol alpha, delta, epsilon, and epsilon*. A comparison of the V-max and K-m for this derivative (PMEApp) and dATP demonstrated that the relative efficiency of the incorporation of this analog into the DNA chain is decreasing in the following order: pol delta similar or equal to pol epsilon similar or equal to pol epsilon(*) > pol alpha. Under the reaction conditions, this incorporation amounted to 4.4 to 0.7% of dAMP molecules. Similar K-m values for PMEApp and dATP in pol epsilon and pol epsilon(*) catalyzed reactions revealed that proteolysis of the enzyme probably does not affect the dNTP binding site. The DNA polymerases tested were inhibited by the reaction product (PMEA terminated DNA chain) with similar K-i/K-m ratios (pol alpha 0.2; pol delta, 0.1; pol epsilon 0.05; and pol epsilon(*), 0.06). The associated 3'-5'-exonuclease activity of pol delta, epsilon, and epsilon* was able to excise PMEA from the 3'-OH end of DNA with a rate one order of magnitude lower than that of the dAMP residue. (C) 1999 Elsevier Science Inc.