METAL ACTIVATION OF SYNTHETIC AND DEGRADATIVE ACTIVITIES OF PHI-29 DNA-POLYMERASE, A MODEL ENZYME FOR PROTEIN-PRIMED DNA-REPLICATION

Analysis of metal activation on the synthetic and degradative activities of phi-29 DNA polymerase was carried out in comparison with T4 DNA polymerase and Escherichia coli DNA polymerase I (Klenow fragment). In the three DNA polymerases studied, both the polymerization and the 3' --> 5' exonuclease activity had clear differences in their metal ion requirements. The results obtained support the existence of independent metal binding sites for the synthetic and degradative activities of phi-29 DNA polymerase, according with the distant location of catalytic domains (N-terminal for the 3' --> 5' exonuclease and C-terminal for DNA polymerization) proposed for both Klenow fragment and phi-29 DNA polymerase. Furthermore, DNA competition experiments using phi-29 DNA polymerase suggested that the main differences observed in the metal usage to activate polymerization may be the consequence of metal-induced changes in the enzyme-DNA interactions, whose strength distinguishes processive and nonprocessive DNA polymerases. Interestingly, the initiation of DNA polymerization using a protein as a primer, a special synthetic activity carried out by phi-29 DNA polymerase, exhibited a strong preference for Mn2+ as metal activator. The molecular basis for this preference is mainly the result of a large increase in the affinity for dATP.