3'-]5' EXONUCLEASE ACTIVE-SITE OF PHI-29 DNA-POLYMERASE - EVIDENCE FAVORING A METAL ION-ASSISTED REACTION-MECHANISM

The residues forming the 3' --> 5' exonuclease active site of phi 29 D NA polymerase, located at the N-terminal conserved moths fro I, fro LI and fro III, have been defined by site-directed mutagenesis (Bernad, A., Blanco, L., Lazaro, J. M., Martin, G., and Salas, M. (1989) Cell 5 9, 219-228; Soengas, M. S., Esteban, J. A., Lazaro, J. M., Bernad, A., Blasco, M. A., Salas, M., and Blanco, L. (1992) EMBO J. 11, 4227-4237 ). To understand their catalytic role, the residual exonuclease activi ty of mutants at these active site residues has been kinetically studi ed. The critical function of residues Asp(12), Glu(14) ASp(66), and As p(169) is supported by a 10(5)-fold reduction in the exonuclease catal ytic rate upon single mutation. Residue Tyr(165) seems to play a secon dary role in the exonuclease reaction based on the 10(2)-10(3)-fold re duced catalytic rate of mutants Y165F and Y165C. Most of the mutants w ere specially active in the presence of Mn2+ ions, which could be indi cative of a direct involvement of these residues in a metal ion-assist ed exonucleolytic reaction. The data obtained strongly suggest that th e 3' --> 5' exonuclease active site of phi 29 DNA polymerase is struct urally and functionally similar to that of the Escherichia coil DNA po lymerase I. In addition, these residues were also very important for t he strand displacement ability of phi 29 DNA polymerase, suggesting a structural overlapping of this activity with the 3' --> 5' exonuclease .