Structures of RB69 DNA polymerase ternary complexes reveal multiple modes of metal ion coordination to correct incoming dNTPs

Abstract

We have observed three different modes of metal ion coordination to the triphosphate tails of all correct dNTPs in the crystal structures of 14 ternary complexes of the RB69 pol triple mutant L561A/S565G/Y567A. These include the classic tridendate coordination to all three α,β and γ phosphates and two novel modes of bidendate metal ion coordination, to α,β phosphates and β,γ phosphates. The bidendate dNTP complexes have not been previously observed in any of the ternary crystal structures of RB69 pol reported to date. We have identified residues D684, E686, and E716 as being specifically involved in α,β bidendate coordination. We also showed that replacement of E686 and D684 with alanine residues reduced the catalytic efficiency by 70 fold and 20 fold respectively. These results provide the first direct evidence that the α,β bidendate coordination complex is a kinetically important intermediate in the nucleotidyl transfer reaction pathway.