title: Biochemical analysis of six genetic variants of error-prone human DNA polymerase ι involved in translesion DNA synthesis.
abstract: 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., N(2)-ethyl(Et)G, O(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, N(2)-EtG, O(6)-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 Mg(2+) (but not with Mn(2+)), coinciding with its steady-state kinetic data showing a ∼10-fold increase in kcat/Km for nucleotide incorporation opposite templates (only with Mg(2+)). 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 kcat/Km for nucleotide incorporation opposite templates either with Mg(2+) or Mn(2+), except for that opposite N(2)-EtG with Mn(2+) (showing a 9-fold increase for dCTP incorporation). The Δ1-25 variant bound DNA 20- to 29-fold more tightly than wild-type (with Mg(2+)), 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 Mn(2+) than with Mg(2+). The results indicate that the R96G variation severely impairs most of the Mg(2+)- and Mn(2+)-dependent TLS abilities of pol ι, whereas the Δ1-25 variation selectively and substantially enhances the Mg(2+)-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.
authors.affiliation: Division of Pharmacology, Department of Molecular Cell Biology, and ‡Department of Physiology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine , Suwon, Gyeonggi-do 440-746, Republic of Korea.
authors.forename: Jinsook
authors.initials: J
authors.lastname: Kim
authors.forename: Insil
authors.initials: I
authors.lastname: Song
authors.forename: Ara
authors.initials: A
authors.lastname: Jo
authors.forename: Joo-Ho
authors.initials: JH
authors.lastname: Shin
authors.forename: Hana
authors.initials: H
authors.lastname: Cho
authors.forename: Robert L
authors.initials: RL
authors.lastname: Eoff
authors.forename: F Peter
authors.initials: FP
authors.lastname: Guengerich
authors.forename: Jeong-Yun
authors.initials: JY
authors.lastname: Choi
mesh_terms: D019943:Amino Acid Substitution; D001483:Base Sequence; D004247:DNA / Q000096:biosynthesis* / Q000737:chemistry; D017931:DNA Primers / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005454:Fluorescence Polarization; D006151:Guanosine / Q000031:analogs & derivatives / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D008345:Manganese / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D000094769:DNA Polymerase iota
chemical_list: D017931:DNA Primers; D011994:Recombinant Proteins; D006151:Guanosine; D008345:Manganese; D004247:DNA; D004259:DNA-Directed DNA Polymerase; D008274:Magnesium; D000094769:DNA Polymerase iota; C000722070:POLI protein, human