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.

Polycyclic aromatic hydrocarbons (PAHs) are significant environmental pollutants representing an important risk factor in human cancers. DNA adducts formed by the ultimate carcinogens of PAHs are potentially toxic, mutagenic and carcinogenic. DNA repair represents an important defense system against these genotoxic insults. Using a human cell-free system we have examined repair of DNA lesions induced by several PAH dihydrodiol epoxides, including anti-(+/-)-benzo[a]pyrene-trans-7,8-dihydrodiol-9,10-epoxide, anti-(+/-)-benz[a]anthracene-trans-3,4-dihydrodiol-1,2-epoxide, anti-(+/-)-benz[a]anthracene-trans-8,9-dihydrodiol-10,11-epoxide, anti-(+/-)-benzo[b]fluoranthene-trans-9,10-dihydrodiol-11,12-epoxide and anti-(+/-)-chrysene-trans-1,2-dihydrodiol-3,4-epoxide. Effective repair of DNA damage induced by these five PAH metabolites was detected. Two distinct mechanisms of excision repair were observed. The major repair mechanism is nucleotide excision repair (NER). The other mechanism is independent of NER and correlated with the presence of apurinic/apyrimidinic sites in the damaged DNA, thus presumably reflecting base excision repair (BER). However, the contribution of BER to different PAH lesions varied in vitro. These results suggest the possibility that BER may also play an important role in repair of certain PAH-induced DNA lesions.

DNA polymerase λ (Pol λ) is a DNA polymerase β (Pol β)-like enzyme with both DNA synthetic and 5'-deoxyribose-5'-phosphate lyase domains. Resent biochemical studies implicated Pol λ as a backup enzyme to Pol ß in the mammalian base excision repair (BER) pathway. To examine the interrelationship between Pol λ and Pol ß in BER of DNA damage in living cells, we disrupted the genes for both enzymes either singly or in combination in the chicken DT40 cell line and then characterized BER phenotypes. Disruption of the genes for both polymerases caused hypersensitivity to H 2 O 2 -induced cytotoxicity, whereas the effect of disruption of either polymerase alone was only modest. Similarly, BER capacity in cells after H 2 O 2 exposure was lower in Pol β −/− /Pol λ −/− cells than in Pol β −/− , wild-type and Pol λ −/− cells, which were equivalent. These results suggest that these polymerases can complement for one another in counteracting oxidative DNA damage. Similar results were obtained in assays for in vitro BER capacity using cell extracts. With MMS-induced cytotoxicity, there was no significant effect on either survival or BER capacity from Pol λ gene disruption. A strong hypersensitivity and reduction in BER capacity was observed for Pol β −/− /Pol λ −/− and Pol β −/− cells, suggesting that Pol β had a dominant role in counteracting alkylation DNA damage in this cell system.

Benzo[a]pyrene is a potent environmental carcinogen, which can be metabolized in cells to the DNA damaging agent anti-benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (anti-BPDE). We hypothesize that mutations induced by BPDE DNA adducts are mainly generated through an error-prone translesion synthesis that requires a specialized DNA polymerase (Pol). Using an in vivo mutagenesis assay in the yeast model system, we have examined the potential roles of Pol(zeta) and Pol(eta) in (+/-)-anti-BPDE-induced mutagenesis. In cells proficient in mutagenesis, (+/-)-anti-BPDE induced 85% base substitutions with predominant G --> C followed by G --> T transversions, 9% deletions of 1-3 nucleotides, and 6% insertions of 1-3 nucleotides. In rad30 mutant cells lacking Pol(eta), (+/-)-anti-BPDE-induced mutagenesis was reduced and accompanied by a moderate decrease in base substitutions and more significant decrease in deletions and insertions of 1-3 nucleotides. In rev3 mutant cells lacking Pol(zeta), (+/-)-anti-BPDE-induced mutagenesis was mostly abolished, leading to a great decrease in both base substitutions and deletions/insertions of 1-3 nucleotides. In contrast, large deletions/insertions were significantly increased in cells lacking Pol(zeta). Consistent with the in vivo results, purified yeast Pol(zeta) performed limited translesion synthesis opposite (+)- and (-)-trans-anti-BPDE-N(2)-dG DNA adducts with predominant G incorporation opposite the lesion. These results show that (+/-)-anti-BPDE-induced mutagenesis in yeast requires Pol(zeta) and partially involves Pol(eta) and suggest that Pol(zeta) directly participates in nucleotide insertions opposite the lesion, while Pol(eta) significantly contributes to deletions and insertions of 1-3 nucleotides.

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The base excision DNA repair (BER) pathway known to occur in Caenorhabditis elegans has not been well characterized. Even less is known about the DNA polymerase (pol) requirement for the gap-filling step during BER. We now report on characterization of in vitro uracil-DNA initiated BER in C. elegans. The results revealed single-nucleotide (SN) gap-filling DNA polymerase activity and complete BER. The gap-filling polymerase activity was not due to a DNA polymerase β (pol β) homolog, or to another X-family polymerase, since computer-based sequence analyses of the C. elegans genome failed to show a match for a pol β-like gene or other X-family polymerases. Activity gel analysis confirmed the absence of pol β in the C. elegans extract. BER gap-filling polymerase activity was partially inhibited by both dideoxynucleotide and aphidicolin. The results are consistent with a combination of both replicative polymerase(s) and lesion bypass/BER polymerase pol θ contributing to the BER gap-filling synthesis. Involvement of pol θ was confirmed in experiments with extract from pol θ null animals. The presence of the SN BER in C. elegans is supported by these results, despite the absence of a pol β-like enzyme or other X-family polymerase.