STRUCTURAL PRINCIPLES FOR THE INHIBITION OF THE 3'-5'-EXONUCLEASE ACTIVITY OF ESCHERICHIA-COLI DNA-POLYMERASE-I BY PHOSPHOROTHIOATES

A two-metal-ion catalytic mechanism has previously been proposed for s everal phosphoryl-transfer enzymes. In order to extend the structural basis of this mechanism, crystal structures of three single-stranded D NA substrates bound to the 3'-5' exonucleolytic active site of the lar ge fragment of DNA polymerase I from Escherichia coli have been elucid ated. The first is a 2.1 Angstrom resolution structure of a Michaelis complex between the large fragment (or Klenow fragment, KF) and a sing le-stranded DNA substrate, stabilized by low pH and flash-freezing. Th e positions and identities of the catalytic metal ions, a Zn2+ at site A and a Mg2+ at site B, have been clearly established. The structural and kinetic consequences of sulfur substitutions in the scissile phos phate have been explored. A complex with the R-p isomer of phosphoroth ioate DNA, refined at 2.2 Angstrom resolution, shows Zn2+ bound to bot h metal sites and a mispositioning of the substrate and attacking nucl eophile. The complex with the Sr Phosphorothioate at 2.3 Angstrom reso lution reveals that metal ions do not bind in the active site, having been displaced by a bulky sulfur atom. Steady-state kinetic experiment s show that catalyzed hydrolysis of the R-p isomer was reduced only ab out 15-fold, while no enzyme activity could be detected with the S-p p hosphorothioate, consistent with the structural observations. Furtherm ore, Mn2+ could not rescue the activity of the exonuclease on the Sp p hosphorothioate. Taken together, these studies confirm and extend the proposed two-metal-ion exonuclease mechanism and provide a structural context to explain the effects of sulfur substitutions on this and oth er phosphoryl-transfer enzymes. These experiments also suggest that th e possibility of metal-ion exclusion be taken into account when interp reting the results of Mn2+ rescue experiments. (C) 1998 Academic Press Limited.