FEN1 Functions in Long Patch Base Excision Repair Under Conditions of Oxidative Stress in Vertebrate Cells

From in vitro studies, flap endonuclease 1 (FEN1) has been proposed to play a role in the long patch (LP) base excision repair (BER) sub-pathway. Yet, the role of FEN1 in BER in the context of the living vertebrate cell has not been thoroughly explored. In the present study, we cloned a DT40 chicken cell line with a deletion in the FEN1 gene and found that these FEN1-deficient cells exhibited hypersensitivity to H2O2. This oxidant produces genotoxic lesions that are repaired by BER, suggesting that the cells have a deficiency in BER affecting survival. In experiments with extracts from the isogenic FEN1 null and wild type cell lines, the LP-BER activity of FEN1 null cells was deficient, whereas repair by the single-nucleotide BER sub-pathway was normal. Other consequences of the FEN1 deficiency also were evaluated. These results illustrate that FEN1 plays a role in LP-BER in higher eukaryotes, presumably by processing the flap-containing intermediates of BER

Interplay between DNA polymerases β and λ in repair of oxidation DNA damage in chicken DT40 cells

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 H2O2-induced cytotoxicity, whereas the effect of disruption of either polymerase alone was only modest. Similarly, BER capacity in cells after H2O2 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

Single-nucleotide base excision repair DNA polymerase activity in C. elegans in the absence of DNA polymerase β

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

Lack of EGF receptor contributes to drug sensitivity of human germline cells

Germline mutations have been associated with generation of various types of tumour. In this study, we investigated genetic alteration of germline tumours that affect the drug sensitivity of cells. Although all germline tumour cells we tested were hypersensitive to DNA-damaging drugs, no significant alteration was observed in their DNA repair activity or the expression of DNA repair proteins. In contrast, germline tumours expressed very low level of epidermal growth factor receptor (EGFR) compared to drug-resistant ovarian cancer cells. An immunohistochemical analysis indicated that most of the primary germline tumours we tested expressed very low level of EGFR. In accordance with this, overexpression of EGFR in germline tumour cells showed an increase in drug resistance, suggesting that a lack of EGFR, at least in part, contributes to the drug sensitivity of germline tumours

Transcriptional Mutagenesis Induced by 8-Oxoguanine in Mammalian Cells

Most of the somatic cells of adult metazoans, including mammals, do not undergo continuous cycles of replication. Instead, they are quiescent and devote most of their metabolic activity to gene expression. The mutagenic consequences of exposure to DNA–damaging agents are well documented, but less is known about the impact of DNA lesions on transcription. To investigate this impact, we developed a luciferase-based expression system. This system consists of two types of construct composed of a DNA template containing an 8-oxoguanine, paired either with a thymine or a cytosine, placed at defined positions along the transcribed strand of the reporter gene. Analyses of luciferase gene expression from the two types of construct showed that efficient but error-prone transcriptional bypass of 8-oxoguanine occurred in vivo, and that this lesion was not repaired by the transcription-coupled repair machinery in mammalian cells. The analysis of luciferase activity expressed from 8OG:T-containing constructs indicated that the magnitude of erroneous transcription events involving 8-oxoguanine depended on the sequence contexts surrounding the lesion. Additionally, sequencing of the transcript population expressed from these constructs showed that RNA polymerase II mostly inserted an adenine opposite to 8-oxoguanine. Analysis of luciferase expression from 8OG:C-containing constructs showed that the generated aberrant mRNAs led to the production of mutant proteins with the potential to induce a long-term phenotypical change. These findings reveal that erroneous transcription over DNA lesions may induce phenotypical changes with the potential to alter the fate of non-replicating cells

Interplay between DNA polymerases beta and lambda in repair of oxidation DNA damage in chicken DT40 cells.

DNA polymerase lambda (Pol lambda) is a DNA polymerase beta (Pol beta)-like enzyme with both DNA synthetic and 5'-deoxyribose-5'-phosphate lyase domains. Recent biochemical studies implicated Pol lambda as a backup enzyme to Pol beta in the mammalian base excision repair (BER) pathway. To examine the interrelationship between Pol lambda and Pol beta 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 beta(-/-)/Pol lambda(-/-) cells than in Pol beta(-/-), wild-type, and Pol lambda(-/-) 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 lambda gene disruption. A strong hypersensitivity and reduction in BER capacity was observed for Pol beta(-/-)/Pol lambda(-/-) and Pol beta(-/-) cells, suggesting that Pol beta had a dominant role in counteracting alkylation DNA damage in this cell system

Vertebrate POLQ and POLß cooperate in base excision repair of oxidative DNA damage.

Base excision repair (BER) plays an essential role in protecting cells from mutagenic base damage caused by oxidative stress, hydrolysis, and environmental factors. POLQ is a DNA polymerase, which appears to be involved in translesion DNA synthesis (TLS) past base damage. We disrupted POLQ, and its homologs HEL308 and POLN in chicken DT40 cells, and also created polq/hel308 and polq/poln double mutants. We found that POLQ-deficient mutants exhibit hypersensitivity to oxidative base damage induced by H2O2, but not to UV or cisplatin. Surprisingly, this phenotype was synergistically increased by concomitant deletion of the major BER polymerase, POLβ. Moreover, extracts from a polq null mutant cell line show reduced BER activity, and POLQ, like POLβ, accumulated rapidly at sites of base damage. Accordingly, POLQ and POLβ share an overlapping function in the repair of oxidative base damage. Taken together, these results suggest a role for vertebrate POLQ in BER. © 2006 Elsevier Inc. All rights reserved