The activity of selected RB69 DNA polymerase mutants can be restored by manganese ions: the existence of alternative metal ion ligands used during the polymerization cycle.
Site specific mutants in the pol active center of RB69 DNA polymerase have been produced and studied using rapid chemical-quench techniques. Pre-steady-state kinetic analysis carried out with Mg(2+) and Mn(2+) has enabled us to divide the mutants into two groups. One group had greatly reduced k(pols) values in the presence of Mg(2+) but responded to Mn(2+) which restored the k(pol) values for the nucleotidyl transfer reaction to near wild-type levels. The other group of mutants also had lower k(pol) values, relative to that of the wild-type polymerase, but could not be rescued by Mn(2+). The behavior of these mutants was interpreted in terms of the crystal structures of the available RB69 pol complexes. Our results on the metal ion dependence of the D621A and E686A mutants, together with knowledge of the position of their side chains in two different RB69 pol conformations, suggest that these acidic residues serve as alternative ligands for the metal ions destined to occupy the A and B catalytic sites. We infer that this occurs prior to the conformational change that produces the ternary RB69 pol complex in which the A and B metal ions are ligated by D623 and D411 as the enzyme is poised for phosphoryl transfer.
Zakharova
E
E
Department of Molecular Biophysics and Biochemistry, Yale University, 333 Cedar Street, New Haven, Connecticut 06520, USA.
Wang
J
J
Konigsberg
W
W
D001665:Binding Sites; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D003848:Deoxyguanine Nucleotides / Q000378:metabolism; D007477:Ions; D007700:Kinetics; D008024:Ligands; D008345:Manganese / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D003848:Deoxyguanine Nucleotides; D007477:Ions; D008024:Ligands; D014764:Viral Proteins; D008345:Manganese; C007257:2'-deoxyguanosine 5'-phosphate; D004259:DNA-Directed DNA Polymerase; C463461:bacteriophage RB69 DNA polymerase