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 Mn²⁺ than Mg²⁺. The human germline R96G variant impairs both Mn²⁺-dependent and Mg²⁺-dependent activities of pol ι, whereas the Δ1-25 variant selectively enhances its Mg²⁺-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 Mn²⁺ (0.15 mM) instead of Mg²⁺ (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 Mg²⁺ when compared with wild type, substantially attenuated by substitution with Mn²⁺. 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 Mg²⁺ or Mn²⁺, revealed that Mn²⁺ achieves more optimal octahedral coordination geometry than Mg²⁺, 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.