GTases are members of the covalent nucleotidyl transferase superfamily, which also includes DNA and RNA ligases [21] . The crystal structures of various family members revealed a common tertiary structure consisting of an N-terminal NT domain and a Cterminal OB-fold domain [22, 23, 53, 54, 55] . Based on both the sequence and structural similarities between GTases and DNA/ RNA ligases (Fig. 6A and B) , it is tempting to speculate that MPA might also be an inhibitor of DNA/RNA ligases. DNA/RNA ligation entails three sequential nucleotidyl transfer steps, and the first two steps are mechanistically related to the GTase reaction [56] . In the first step of the ligation reaction, nucleophilic attack on the a-phosphorus of ATP by ligase results in the formation of a covalent ligase-adenylate intermediate with the concomitant release of pyrophosphate. In the second step, AMP is transferred to the 59-end of the 59-phosphate-terminated DNA strand to form a DNA-adenylate complex. In the last step of the reaction, The the polynucleotides are joined with the concomitant release of AMP [56] . To verify the potential ability of MPA to inhibit DNA/RNA ligases, we have monitored the effect of MPA on the purified DNA ligase encoded by Chlorella virus. The ability of MPA to inhibit the complete ligation reaction was monitored by incubating the purified enzyme with ATP and a DNA duplex containing a centrally located nick. The reaction products were then analyzed on a denaturing polyacrylamide gel. As can be seen in Fig. 7A , the presence of increasing concentrations of MPA significanlty decreased strand joining by the Chlorella virus DNA ligase. MPA was shown to inhibit the strand-joining activity by 50% at 700 mM. We next set out to investigate which of the first two catalytic steps of the ligation reaction is inhibited by MPA. The first step of the reaction, i.e. the formation of the enzyme-AMP covalent intermediate, was monitored by incubating the purified enzyme in the presence of [alpha-32 P]ATP. The radiolabeled covalent enzyme-AMP complex was then visualized by autoradiography following electrophoresis on a denaturant polyacrylamide gel. Similarly to what was observed in the case of the GTase activity, our results indicate that very high concentrations of MPA are required to inhibit the first step of the reaction, i.e. the formation of the enzyme-AMP covalent complex (Fig. 7B) . We determined that a concentration of ,3.0 mM of MPA is required to inhibit the first step of the ligation reaction by 50%, a concentration that corresponds to 4times the amount required to inhibit the overall reaction. The effect of MPA on the second step of the ligation reaction (i.e. the transfer of the AMP from ligase-adenylate to a 59-phosphate terminus acceptor DNA) was next evaluated. In a typical ligation reaction, the adenylate-DNA intermediate would not be detected since the enzyme is highly efficient in ligating the two DNA strands [57] . However, as reported previously [58] , the adenylate-DNA intermediate can accumulate to high levels when the enzyme acts on a substrate that contains a 1-nt gap between the reactive 39-OH and 59-phosphate strands. Using such an approach, the incubation of the enzyme with ATP and a gapped DNA substrate resulted in the conversion of the 59-radiolabeled 18-mer strand into an adenylated species (AppDNA) that migrated 1-nt slower than the input 18-mer during polyacrylamide gel electrophoresis (Fig. 7C) . The ability of MPA to inhibit this second step of the ligase reaction was investigated by pre-incubating the enzyme with ATP to ensure formation of the covalent enzyme-AMP complex, and by incubating the complex in the presence of the ligation substrate that contains a 1-nt gap between the reactive 39-OH and 59phosphate strands. Using this strategy, it was determined that a concentration of 600 mM of MPA is sufficient to inhibit 50% of the second step of the ligation reaction (Fig. 7C) . Taken together, these results indicate that MPA inhibits the ligation reaction mainly through inhibition of the second step of the reaction, i.e. the catalytic transfer of the AMP moiety onto the 59-phosphate terminus of the nicked DNA substrate.