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

摘要

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., N²-ethyl(Et)G, O⁶-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, N²-EtG, O⁶-MeG, 8-oxoG, or abasic site. The Δ1–25 variant, which is the N-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 kcat/Km 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 kineticdata displaying 5- to 72-fold decreases in kcat/Km for nucleotide incorporationopposite templates either with Mg²⁺ or Mn²⁺,except for that opposite N²-EtG with Mn²⁺ (showing a 9-fold increase for dCTP incorporation). TheΔ1–25 variant bound DNA 20- to 29-fold more tightly thanwild-type (with Mg²⁺), but the R96G variant bound DNA 2-foldless tightly than wild-type. The DNA-binding affinity of wild-type,but not of the Δ1–25 variant, was ∼7-fold strongerwith 0.15 mM Mn²⁺ than with Mg²⁺. The resultsindicate 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 substantiallyenhances the Mg²⁺-dependent TLS capability of pol ι,emphasizing the potential translational importance of these pol ιgenetic variations, e.g., individual differences in TLS, mutation,and cancer susceptibility to genotoxic carcinogens.