The oxidatively induced DNA lesions 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG) and 4,6-diamino-5-formamidopyrimidine (FapyA) are formed abundantly in DNA of cultured cells or tissues exposed to ionizing radiation or to other free radical-generating systems. In vitro studies indicate that these lesions are miscoding, can block the progression of DNA polymerases, and are substrates for base excision repair. However, no study has yet addressed how these lesions are metabolized in cellular extracts. The synthesis of oligonucleotides containing FapyG and FapyA at defined positions was recently reported. These constructs allowed us to investigate the repair of Fapy lesions in nuclear and mitochondrial extracts from wild type and knock-out mice lacking the two major DNA glycosylases for repair of oxidative DNA damage, OGG1 and NTH1. The background level of FapyG/FapyA in DNA from these mice was also determined. Endogenous FapyG levels in liver DNA from wild type mice were significantly higher than 8-hydroxyguanine levels. FapyG and FapyA were efficiently repaired in nuclear and mitochondrial extracts from wild type animals but not in the glycosylasedeficient mice. Our results indicated that OGG1 and NTH1 are the major DNA glycosylases for the removal of FapyG and FapyA, respectively. Tissue-specific analysis suggested that other DNA glycosylases may contribute to FapyA repair when NTH1 is poorly expressed. We identified NEIL1 in liver mitochondria, which could account for the residual incision activity in the absence of OGG1 and NTH1. FapyG and FapyA levels were significantly elevated in DNA from the knock-out mice, underscoring the biological role of OGG1 and NTH1 in the repair of these lesions.A large number of DNA base modifications are formed by oxidative damage to DNA (for review, see Ref. 1). Some of these lesions are generated at high rates, even in the absence of exogenous DNA-damaging agents. For instance, it was estimated that 100 -500 8-hydroxyguanines 3 are formed per day in a human cell (2). 8-oxoG is one of the most studied DNA lesions, and it is often used as a biomarker of oxidative DNA damage. However, ring-opened formamidopyrimidine lesions, 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG) and 4,6-diamino-5-formamidopyrimidine (FapyA), are formed at equal or higher levels than 8-oxoG after oxidative stress (3-5). These lesions result from hydroxyl radical attack on guanine and adenine, respectively, followed by one-electron reduction of the hydroxyl adduct radicals (1), which are also intermediates in the formation of 8-oxoG and 8-oxoA (6). Formation of Fapy lesions in DNA upon UV radiation has also been reported (7). FapyA (8) and FapyG (9) are miscoding in vitro, both directing the preferential misincorporation of adenine opposite the lesions by a bacterial DNA polymerase (Klenow exo Ϫ ). Experiments using the methylated analogue of FapyG, i.e. 2,6-diamino-4-hydroxy-5-Nmethylformamidopyrimidine (Me-FapyG), have suggested that formamidopyrimidines might also constitute blocks to DNA poly...

Oligodeoxynucleotides containing formamidopyrimidine lesions and C-nucleoside analogues at defined sites were prepared by solid-phase synthesis and in some cases enzymatic ligation. Formamidopyrimidine lesions were introduced as dinucleotides to prevent rearrangement to their pyranose isomers. Oligodeoxynucleotides containing single diastereomers of C-nucleoside analogues of Fapy.dA were introduced by using the respective phosphoramidites. The formamidopyrimidine lesions reduce the T(M) of dodecamers relative to their unmodified nucleotide counterparts when opposite the nucleotide proper base-pairing partner. However, duplexes containing Fapy.dG-dA mispairs melt significantly higher than those comprised of dG-dA. All duplexes containing Fapy.dA-dX or its C-nucleoside analogue melt lower than the respective complexes containing dA-dX. Studies of the alkaline lability of oligodeoxynucleotides containing formamidopyrimidine lesions indicate that Fapy.dA is readily identified as an alkali-labile lesion with use of piperidine (1.0 M, 90 degrees C, 20 min), but Fapy.dG is less easily identified in this manner.

Under conditions of oxidative stress, the formamidopyrimidine lesions (FapyG and FapyA) are formed in competition with the corresponding 8-oxopurines (OG and OA) from a common intermediate. In order to reveal features of the repair of these lesions, and the potential contribution of repair in mitigating or exacerbating the mutagenic properties of Fapy lesions, their excision by three glycosylases, Fpg, hOGG1 and Ntg1, was examined in various base pair contexts under single-turnover conditions. FapyG was removed at least as efficiently as OG by all three glycosylases. In addition, the rates of removal of FapyG by Fpg and hOGG1 were influenced by their base pair partner, with preference for removal when base paired with the correct Watson-Crick partner C. With the FapyA lesion, Fpg and Ntg1 catalyze its removal more readily than OG opposite all four natural bases. In contrast, the removal of FapyA by hOGG1 was not as robust as FapyG or OG, and was only significant when the lesion was paired with C. The discrimination by the various glycosylases with respect to the opposing base was highly dependent on the identity of the lesion. OG induced the greatest selectivity against its removal when part of a promutagenic base pair. The superb activity of the various OG glycosylases toward removal of FapyG and FapyA in vitro suggests that these enzymes may act upon these oxidized lesions in vivo. The differences in the activity of the various glycosylases for removal of FapyG and FapyA compared to OG in nonmutagenic versus promutagenic base pair contexts may serve to alter the mutagenic profiles of these lesions in vivo.

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The antiviral drug 2,3-didehydro-3-deoxythymidine (D4T; also know as stavudine and Zerit), which is used against human immunodeficiency virus (HIV), causes delayed toxicity (peripheral neuropathy) in long-term use. After examining a series of 2,3-didehydro-3-deoxy-4-substituted thymidine (4-substituted D4T) analogs, 4-ethynyl D4T was found to have a fivefold-better antiviral effect and to cause less cellular and mitochondrial toxicity than D4T. The antiviral activity of this compound can be reversed by dThd but not by dCyd. The compound acted synergistically with ␤-L-2,3-deoxy-3-thiacytidine (also known as lamivudine) and ␤-L-2,3-dideoxy-2,3-didehydro-5-fluorocytidine (also known as elvucitabine) and additively with 2,3-dideoxyinosine (also known as didanosine and Videx) and 3-azido-3-deoxythymidine (also known as Retovir and zidovudine) against HIV. 4-Ethynyl D4T is phosphorylated by purified human thymidine kinase 1 (TK-1) from CEM cells with a faster relative V max and a lower K m value than D4T. The efficiency of TK-1 in the phosphorylation of 4-ethynyl D4T is fourfold better than that of D4T. While D4T is broken down by the catabolic enzyme thymidine phosphorylase, the level of breakdown of 4-ethynyl D4T was below detection. Since 4-ethynyl D4T has increased anti-HIV activity and decreased toxicity and interacts favorably with other currently used anti-HIV drugs, it should be considered for further development as an anti-HIV drug.