Mammalian DNA polymerase (pol) is a member of the X-family of DNA polymerases and has striking enzymatic and structural similarities to mammalian DNA pol ␤. Because pol ␤ provides two important enzymatic activities for base excision repair (BER), we examined whether pol might also contribute to BER. We used extracts from mouse embryonic fibroblasts representing wild-type and null genotypes for pol ␤ and pol . In combination with neutralizing antibodies against pol ␤ and pol , our results show a BER deficiency in the pol ؊/؊ cell extract compared with extract from isogenic wild-type cells. In addition, the pol antibody strongly reduced in vitro BER in the pol ␤ ؊/؊ cell extract. These data indicate that pol is able to contribute to BER in mouse fibroblast cell extract.Cells are constantly exposed to environmental stress agents, endogenous reactive oxygen species and alkylating molecules, and other reactive metabolites that are capable of modifying DNA. Cells have several mechanisms by which they protect themselves from the detrimental effects of genotoxic compounds. Base excision repair (BER) 1 is the predominant DNA repair system in mammalian cells for eliminating discrete DNA base lesions (1). Single-nucleotide BER, one subpathway of BER, results in replacement of only the modified nucleotide and is initiated by a lesion-specific DNA glycosylase. Monofunctional DNA glycosylases hydrolyze the glycosidic bond between the sugar and base of the damaged nucleotide, whereas bifunctional DNA glycosylases excise the damaged base and cleave the phosphodiester backbone 3Ј to the resulting abasic site. In the former case, the resulting apurinic/apyrimidinic site is cleaved by apurinic/apyrimidinic endonuclease (APE), producing a single-strand DNA break. DNA polymerase gap-filling DNA synthesis and 5Ј-deoxyribose phosphate (dRP) removal generates the substrate for the final BER step, DNA ligation. In higher organisms, DNA polymerase (pol)

DNA polymerase (pol ) is a member of the X family of DNA polymerases that has been implicated in both base excision repair and non-homologous end joining through in vitro studies. However, to date, no phenotype has been associated with cells deficient in this DNA polymerase. Here we show that pol null mouse fibroblasts are hypersensitive to oxidative DNA damaging agents, suggesting a role of pol in protection of cells against the cytotoxic effects of oxidized DNA. Additionally, pol co-immunoprecipitates with an oxidized base DNA glycosylase, single-strand-selective monofunctional uracil-DNA glycosylase (SMUG1), and localizes to oxidative DNA lesions in situ. From these data, we conclude that pol protects cells against oxidative stress and suggest that it participates in oxidative DNA damage base excision repair.

Base excision repair (BER) is a DNA repair pathway designed to correct small base lesions in genomic DNA. While DNA polymerase beta (pol β) is known to be the main polymerase in the BER pathway, various studies have implicated other DNA polymerases in back-up roles. One such polymerase, DNA polymerase lambda (pol λ), was shown to be important in BER of oxidative DNA damage. To further explore roles of the X-family DNA polymerases λ and β in BER, we prepared a mouse embryonic fibroblast cell line with deletions in the genes for both pol β and pol λ. Neutral red viability assays demonstrated that pol λ and pol β double null cells were hypersensitive to alkylating and oxidizing DNA damaging agents. In vitro BER assays revealed a modest contribution of pol λ to single-nucleotide BER of base lesions. Additionally, using co-immunoprecipitation experiments with purified enzymes and whole cell extracts, we found that both pol λ and pol β interact with the upstream DNA glycosylases for repair of alkylated and oxidized DNA bases. Such interactions could be important in coordinating roles of these polymerases during BER.