Biochemical Analysis of Six Genetic Variants of Error-Prone Human DNA Polymerase ι Involved in Translesion DNA Synthesis

DNA polymerase (pol) ι is the most error-prone among the Y-family polymerases that participate in translesion synthesis (TLS). Pol ι can bypass various DNA lesions, e.g., <i>N</i>²-ethyl­(Et)­G, <i>O</i>⁶-methyl­(Me)­G, 8-oxo-7,8-dihydroguanine (8-oxoG), and an abasic site, though frequently with low fidelity. We assessed the biochemical effects of six reported genetic variations of human pol ι on its TLS properties, using the recombinant pol ι (residues 1–445) proteins and DNA templates containing a G, <i>N</i>²-EtG, <i>O</i>⁶-MeG, 8-oxoG, or abasic site. The Δ1–25 variant, which is the <i>N</i>-terminal truncation of 25 residues resulting from an initiation codon variant (c.3G > A) and also is the formerly misassigned wild-type, exhibited considerably higher polymerase activity than wild-type with Mg²⁺ (but not with Mn²⁺), coinciding with its steady-state kinetic data showing a ∼10-fold increase in <i>k</i>_cat/<i>K</i>_m for nucleotide incorporation opposite templates (only with Mg²⁺). The R96G variant, which lacks a R96 residue known to interact with the incoming nucleotide, lost much of its polymerase activity, consistent with the kinetic data displaying 5- to 72-fold decreases in <i>k</i>_cat/<i>K</i>_m for nucleotide incorporation opposite templates either with Mg²⁺ or Mn²⁺, except for that opposite <i>N</i>²-EtG with Mn²⁺ (showing a 9-fold increase for dCTP incorporation). The Δ1–25 variant bound DNA 20- to 29-fold more tightly than wild-type (with Mg²⁺), but the R96G variant bound DNA 2-fold less tightly than wild-type. The DNA-binding affinity of wild-type, but not of the Δ1–25 variant, was ∼7-fold stronger with 0.15 mM Mn²⁺ than with Mg²⁺. The results indicate that the R96G variation severely impairs most of the Mg²⁺- and Mn²⁺-dependent TLS abilities of pol ι, whereas the Δ1–25 variation selectively and substantially enhances the Mg²⁺-dependent TLS capability of pol ι, emphasizing the potential translational importance of these pol ι genetic variations, e.g., individual differences in TLS, mutation, and cancer susceptibility to genotoxic carcinogens