Mitochondrial aprataxin (APTX) protects the mitochondrial genome from the consequence of ligase failure by removing the abortive ligation product, i.e. the 5′-adenylate (5′-AMP) group, during DNA replication and repair. In the absence of APTX activity, blocked base excision repair (BER) intermediates containing the 5′-AMP or 5′-adenylated-deoxyribose phosphate (5′-AMP-dRP) lesions may accumulate. In the current study, we examined DNA polymerase (pol) γ and pol β as possible complementing enzymes in the case of APTX deficiency. The activities of pol β lyase and FEN1 nucleotide excision were able to remove the 5′-AMP-dRP group in mitochondrial extracts from APTX−/− cells. However, the lyase activity of purified pol γ was weak against the 5′-AMP-dRP block in a model BER substrate, and this activity was not able to complement APTX deficiency in mitochondrial extracts from APTX−/−Pol β−/− cells. FEN1 also failed to provide excision of the 5′-adenylated BER intermediate in mitochondrial extracts. These results illustrate the potential role of pol β in complementing APTX deficiency in mitochondria.

Chemotherapeutic nucleoside analogs, such as Ara-C, 5-Fluorouracil (5-FU) and Trifluridine (FTD), are frequently incorporated into DNA by the replicative DNA polymerases. However, it remains unclear how this incorporation kills cycling cells. There are two possibilities: Nucleoside analog triphosphates inhibit the replicative DNA polymerases, and/or nucleotide analogs mis-incorporated into genomic DNA interfere with the next round of DNA synthesis as replicative DNA polymerases recognize them as template DNA lesions, arresting synthesis. To address the first possibility, we selectively disrupted the proofreading exonuclease activity of DNA polymerase ε (Polε), the leading-strand replicative polymerase in avian DT40 and human TK6 cell lines. To address the second, we disrupted RAD18, a gene involved in translesion DNA synthesis, a mechanism that relieves stalled replication. Strikingly, POLE1exo−/− cells, but not RAD18−/− cells, were hypersensitive to Ara-C, while RAD18−/− cells were hypersensitive to FTD. gH2AX focus formation following a pulse of Ara-C was immediate and did not progress into the next round of replication, while gH2AX focus formation following a pulse of 5-FU and FTD was delayed to the next round of replication. Biochemical studies indicate that human proofreading-deficient Polε-exo− holoenzyme incorporates Ara-CTP, but subsequently extend from this base several times less efficiently than from intact nucleotides. Together our results suggest that Ara-C acts by blocking extension of the nascent DNA strand and is counteracted by the proofreading activity of Polε, while 5-FU and FTD are efficiently incorporated but act as replication fork blocks in the subsequent S phase, which is counteracted by translesion synthesis.

Metformin is a biguanide drug that is widely used in the treatment of diabetes. Epidemiological studies have indicated that metformin exhibits anti-cancer activity. However, the molecular mechanisms underlying this activity currently remain unclear. We hypothesized that metformin is cytotoxic in a tumor-specific environment such as glucose deprivation and/or low oxygen (O2) tension. We herein demonstrated that metformin was highly cytotoxic under glucose-depleted, but not hypoxic (2% O2) conditions. In order to elucidate the underlying mechanisms of this selective cytotoxicity, we treated exposed DNA repair-deficient chicken DT40 cells with metformin under glucose-depleted conditions and measured cellular sensitivity. Under glucose-depleted conditions, metformin specifically killed fancc and fancl cells that were deficient in FANCC and FANCL proteins, respectively, which are involved in DNA interstrand cross-link repair. An analysis of chromosomal aberrations in mitotic chromosome spreads revealed that a clinically relevant concentration of metformin induced DNA double-strand breaks (DSBs) in fancc and fancl cells under glucose-depleted conditions. In summary, metformin induced DNA damage under glucose-depleted conditions and selectively killed cells. This metformin-mediated selective toxicity may suppress the growth of malignant tumors that are intrinsically deprived of glucose.