title: Two distinct translesion synthesis pathways across a lipid peroxidation-derived DNA adduct in mammalian cells.
abstract: Translesion DNA synthesis (TLS) of damaged DNA templates is catalyzed by specialized DNA polymerases. To probe the cellular TLS mechanism, a host-vector system consisting of mouse fibroblasts and a replicating plasmid bearing a single DNA adduct was developed. This system was used to explore the TLS mechanism of a heptanone-etheno-dC (H-epsilondC) adduct, an endogenous lesion produced by lipid peroxidation. In wild-type cells, H-epsilondC almost exclusively directed incorporation of dT and dA. Whereas knockout of the Y family TLS polymerase genes, Polh, Polk, or Poli, did not qualitatively affect these TLS events, inactivation of the Rev3 gene coding for a subunit of polymerase zeta or of the Rev1 gene abolished TLS associated with dA, but not dT, insertion. The analysis of results of the cellular studies and in vitro TLS studies using purified polymerases has revealed that the insertion of dA and dT was catalyzed by different polymerases in cells. While insertion of dT can be catalyzed by polymerase eta, kappa, and iota, insertion of dA is catalyzed by an unidentified polymerase that cannot catalyze extension from the resulting dA terminus. Therefore, the extension from this terminus requires the activity of polymerase zeta-REV1. These results provide new insight into how cells use different TLS pathways to overcome a synthesis block.
authors.affiliation: Laboratory of Chemical Biology, Department of Pharmacological Sciences, State University of New York, Stony Brook, New York 11794, Department of Toxicogenetics, Leiden University Medical Center, 2300 RC Leiden, Netherlands, and the Centers for Cancer Pharmacology and Excellence in Environmental Toxicology, University of Pennsylvania, Philadelphia, Pennsylvania 19104.
authors.forename: In-Young
authors.initials: IY
authors.lastname: Yang
authors.affiliation: Laboratory of Chemical Biology, Department of Pharmacological Sciences, State University of New York, Stony Brook, New York 11794, Department of Toxicogenetics, Leiden University Medical Center, 2300 RC Leiden, Netherlands, and the Centers for Cancer Pharmacology and Excellence in Environmental Toxicology, University of Pennsylvania, Philadelphia, Pennsylvania 19104.
authors.forename: Keiji
authors.initials: K
authors.lastname: Hashimoto
authors.affiliation: Laboratory of Chemical Biology, Department of Pharmacological Sciences, State University of New York, Stony Brook, New York 11794, Department of Toxicogenetics, Leiden University Medical Center, 2300 RC Leiden, Netherlands, and the Centers for Cancer Pharmacology and Excellence in Environmental Toxicology, University of Pennsylvania, Philadelphia, Pennsylvania 19104.
authors.forename: Niels
authors.initials: N
authors.lastname: de Wind
authors.affiliation: Laboratory of Chemical Biology, Department of Pharmacological Sciences, State University of New York, Stony Brook, New York 11794, Department of Toxicogenetics, Leiden University Medical Center, 2300 RC Leiden, Netherlands, and the Centers for Cancer Pharmacology and Excellence in Environmental Toxicology, University of Pennsylvania, Philadelphia, Pennsylvania 19104.
authors.forename: Ian A
authors.initials: IA
authors.lastname: Blair
authors.affiliation: Laboratory of Chemical Biology, Department of Pharmacological Sciences, State University of New York, Stony Brook, New York 11794, Department of Toxicogenetics, Leiden University Medical Center, 2300 RC Leiden, Netherlands, and the Centers for Cancer Pharmacology and Excellence in Environmental Toxicology, University of Pennsylvania, Philadelphia, Pennsylvania 19104. Electronic address: maki@pharm.stonybrook.edu.
authors.forename: Masaaki
authors.initials: M
authors.lastname: Moriya
mesh_terms: D000818:Animals; D002461:Cell Line, Transformed; D018736:DNA Adducts / Q000235:genetics / Q000378:metabolism*; D004249:DNA Damage; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D015227:Lipid Peroxidation; D051379:Mice; D000099270:Y-Family DNA Polymerases
chemical_list: D018736:DNA Adducts; D004259:DNA-Directed DNA Polymerase; C486862:REV3L protein, mouse; C498613:Polk protein, mouse; D000099270:Y-Family DNA Polymerases; C112338:Rad30 protein