Kinetic and Structural Impact of Metal Ions and Genetic Variations on Human DNA Polymerase ι.

DNA polymerase (pol) ι is a Y-family polymerase involved in translesion synthesis, exhibiting higher catalytic activity with Mn(2+) than Mg(2+) The human germline R96G variant impairs both Mn(2+)-dependent and Mg(2+)-dependent activities of pol ι, whereas the Δ1-25 variant selectively enhances its Mg(2+)-dependent activity. We analyzed pre-steady-state kinetic and structural effects of these two metal ions and genetic variations on pol ι using pol ι core (residues 1-445) proteins. The presence of Mn(2+) (0.15 mm) instead of Mg(2+) (2 mm) caused a 770-fold increase in efficiency (k(pol)/K(d)(,dCTP)) of pol ι for dCTP insertion opposite G, mainly due to a 450-fold decrease in K(d)(,dCTP) The R96G and Δ1-25 variants displayed a 53-fold decrease and a 3-fold increase, respectively, in k(pol)/K(d)(,dCTP) for dCTP insertion opposite G with Mg(2+) when compared with wild type, substantially attenuated by substitution with Mn(2+) Crystal structures of pol ι ternary complexes, including the primer terminus 3'-OH and a non-hydrolyzable dCTP analogue opposite G with the active-site Mg(2+) or Mn(2+), revealed that Mn(2+) achieves more optimal octahedral coordination geometry than Mg(2+), with lower values in average coordination distance geometry in the catalytic metal A-site. Crystal structures of R96G revealed the loss of three H-bonds of residues Gly-96 and Tyr-93 with an incoming dNTP, due to the lack of an arginine, as well as a destabilized Tyr-93 side chain secondary to the loss of a cation-π interaction between both side chains. These results provide a mechanistic basis for alteration in pol ι catalytic function with coordinating metals and genetic variation.