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-⑀dC) adduct, an endogenous lesion produced by lipid peroxidation. In wild-type cells, H-⑀dC 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 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 , , and , 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 -REV1. These results provide new insight into how cells use different TLS pathways to overcome a synthesis block.Endogenous reactive chemicals such as reactive oxygen species and certain lipid peroxidation products are thought to contribute significantly to aging, age-related degenerative diseases, and cancer (1-4). Cellular DNA is one of their targets, and replication of un-repaired DNA damage introduces mutations into the genome, thereby contributing to the aforementioned biological effects. Heptanone-etheno-dC (H-⑀dC) 2 (Fig. 1) is one of the substituted etheno-base DNA adducts generated by 4-oxo-2(E)-nonenal, a bifunctional electrophilic lipid peroxidation product derived from both arachidonic acid and linoleic acid (5-8). This adduct, as well as H-⑀dG, serves as a biomarker of lipid peroxidation-mediated DNA damage (5). H-⑀dC and H-⑀dG were both found in the DNA of polyps from a cyclooxygenase-2 up-regulated Min mouse, a colorectal cancer mouse model (9). Our previous study has shown that H-⑀dC blocks DNA synthesis and highly miscodes in human cells (10), raising the possibility that cyclooxygenase-2-mediated lipid peroxidation contributes to colorectal carcinogenesis in Min mice through the formation of DNA adducts. Due to its strong genotoxicity and physiological significance, H-⑀dC is an attractive DNA lesion for the mechanistic study of mammalian translesion DNA synthesis (TLS).Recently, it was revealed that various specialized DNA polymerases are actively engaged in DNA synthesis across a DNA lesion (11,12). The catalytic sites of these polymerases are much more spacious than those of replicative polymerases so that they can accommodate a modified template base and an incoming nucleotide (13-15). Accordingly, their fidelity of DNA synthesis is compromised on undamaged template DNA (16 -19). These specialized polyme...

Ablation of XP-V gene causes adipose tissue senescence and metabolic abnormalities

“…Medium or severe DNA damage induces cellular senescence or cell death (apoptosis), which also depends on the tissue type. Our results support previous studies that have shown pol η bypasses oxidative DNA lesions (13,14) because pol η −/− mice exhibit an increased amount of DNA lesions and elevated DNA damage responses. The increase in SA-β-gal activity; up-regulation of p53, p16, and p21; and increased SASP circulating and cellular levels of IL-6 and TNF-α that occur at a very early age in pol η −/− mice provide evidence for a causative role of adipocyte senescence in obesity and the development of insulin resistance.…”

“…Adipose tissue is the main site for lipid peroxidation and acts as a major "sink" to store and handle free radicalinduced peroxidation products (36). Oxidative DNA lesions produced by lipid peroxidation products block DNA replicative polymerases and stall the progression of DNA replication (13,14), which triggers cell cycle arrest and leads to different cellular responses, depending on the magnitude of DNA damage. Mild DNA damage causes temporary pausing of the cell cycle to allow the DNA repair system to repair the damage.…”

“…Pol η has the ability to handle various oxidative DNA lesions, including lipid peroxidation products that impede the progression of DNA replication (13,14). Both pol η and p53 are required for recovery from DNA damage-stalled DNA replication forks (27).…”

“…The expression of pol η correlates with the effectiveness of anticancer therapeutic agents that exert their activity by introducing DNA lesions that block the progression of DNA replication (11). In addition to exogenous introduced DNA lesions, pol η contributes to genomic stability during unperturbed DNA replication (12) and replicates across reactive oxygen species (ROS)-induced oxidative DNA lesions 8-oxoG (7,8-dihydro-8-oxoguanine), thymine glycol, and lipid peroxidation DNA adducts generated during endogenous metabolic processes (13,14). ROS generated from endogenous metabolism or exogenous sources has been associated with cancer, aging, and the metabolic syndrome.…”

The Vital Role of Polymerase ζ and REV1 in Mutagenic, but Not Correct, DNA Synthesis across Benzo[a]pyrene-dG and Recruitment of Polymerase ζ by REV1 to Replication-stalled Site

“…In wild-type MEFs, H-⑀dC was highly miscoding by directing the insertion of dTMP or dAMP (Fig. 2B and supplemental Table S1) as previously reported (30,37). In TKO MEFs, TLS efficiency dropped to 21% of that in wild-type MEFs, indicating that the TLS across H-⑀dC depends on pol , pol , and/or pol .…”