Two Distinct Translesion Synthesis Pathways across a Lipid Peroxidation-derived DNA Adduct in Mammalian Cells

• Published in: The Journal of biological chemistry Vol. 284; no. 1; pp. 191 - 198
• Main Authors: Yang, In-Young, Hashimoto, Keiji, de Wind, Niels, Blair, Ian A., Moriya, Masaaki
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 02.01.2009; American Society for Biochemistry and Molecular Biology
• Subjects: Animals; Cell Line, Transformed; DNA Adducts - genetics; DNA Adducts - metabolism; DNA Damage; DNA-Directed DNA Polymerase - genetics; DNA-Directed DNA Polymerase - metabolism; DNA: , Repair, Recombination, and Chromosome Dynamics; Gene Knockdown Techniques; Lipid Peroxidation; Mice
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M806414200

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-εdC) adduct, an endogenous lesion produced by lipid peroxidation. In wild-type cells, H-εdC 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 ζ 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 η, κ, and ι, 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 ζ-REV1. These results provide new insight into how cells use different TLS pathways to overcome a synthesis block.