Kazuhiro Haraguchi

Repair of Formamidopyrimidines in DNA Involves Different Glycosylases ROLE OF THE OGG1, NTH1, AND NEIL1 ENZYMES

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 glycosylase-deficient 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.

Synthesis and anti-HIV activity of 4'-substituted 4'-thiothymidines: a new entry based on nucleophilic substitution of the 4'-acetoxy group.

Diacetoxylation of 1-(2,5-dideoxy-beta-L-glycero-pent-4-eno-4-thiofuranosyl)thymine (13) with Pb(OAc) 4 allowed introduction of an acetoxy leaving group to the 4-position. Nucleophilic substitution of the resulting 4-acetoxy derivative (14) with silicon reagents enabled us to prepare the 4-phenylthio (17a), 4-azido (18a), 4-methoxy (20a), and 4-allyl (21a) analogues of 4-thiothymidine. 4-Cyano ( 25a) and 4-ethynyl (31) nucleosides were also synthesized from 3,5-bis-O-TBDMS derivative (24). Among novel 4-substituted 4-thiothymidines, the 4-azido (33), 4-cyano (36), and 4-ethynyl (37) derivatives were found to show potent inhibitory activity against HIV-1 and HIV-2. It is noteworthy that 36 and 37 were also inhibitory against replication of HIV variant resistant to 3TC (HIV-1 M184V), being as potent as against HIV-1 IIIB.

Excision of formamidopyrimidine lesions by endonucleases III and VIII is not a major DNA repair pathway in Escherichia coli

Proper maintenance of the genome is of great importance. Consequently, damaged nucleotides are repaired through redundant pathways. We considered whether the genome is protected from formamidopyrimidine nucleosides (Fapy•dA, Fapy•dG) via a pathway distinct from the Escherichia coli guanine oxidation system. The formamidopyrimidines are produced in significant quantities in DNA as a result of oxidative stress and are efficiently excised by formamidopyrimidine DNA glycosylase. Previous reports suggest that the formamidopyrimidine nucleosides are substrates for endonucleases III and VIII, enzymes that are typically associated with pyrimidine lesion repair in E.coli. We investigated the possibility that Endo III and/or Endo VIII play a role in formamidopyrimidine nucleoside repair by examining Fapy•dA and Fapy•dG excision opposite all four native 2′-deoxyribonucleotides. Endo VIII excises both lesions more efficiently than does Endo III, but the enzymes exhibit similar selectivity with respect to their action on duplexes containing the formamidopyrimidines opposite native deoxyribonucleotides. Fapy•dA is removed more rapidly than Fapy•dG, and duplexes containing purine nucleotides opposite the lesions are superior substrates compared with those containing formamidopyrimidine–pyrimidine base pairs. This dependence upon opposing nucleotide indicates that Endo III and Endo VIII do not serve as back up enzymes to formamidopyrimidine DNA glycosylase in the repair of formamidopyrimidines. When considered in conjunction with cellular studies [J. O. Blaisdell, Z. Hatahet and S. S. Wallace (1999) J. Bacteriol., 181, 6396–6402], these results also suggest that Endo III and Endo VIII do not protect E.coli against possible mutations attributable to formamidopyrimidine lesions.

Unlike Catalyzing Error-Free Bypass of 8-OxodGuo, DNA Polymerase λ Is Responsible for a Significant Part of Fapy·dG-Induced G → T Mutations in Human Cells

8-OxodGuo and Fapy·dG induced 10-22% mutations, predominantly G → T transversions, in human embryonic kidney 293T cells in four TG*N sequence contexts, where N = C, G, A, or T. siRNA knockdown of pol λ resulted in 34 and 55% increases in the level of mutations in the progeny from the 8-oxodGuo construct in the TG*T and TG*G sequences, respectively, suggesting that pol λ is involved in error-free bypass of 8-oxodGuo. For Fapy·dG, in contrast, the level of G → T mutations was reduced by 27 and 46% in the TG*T and TG*G sequences, respectively, suggesting that pol λ is responsible for a significant fraction of Fapy·dG-induced G → T mutations.

An alternative synthetic method for 4'-C-ethynylstavudine by means of nucleophilic substitution of 4'-benzoyloxythymine nucleoside.

For the synthesis of 2′,3′ -didehydro-3′ -deoxy-4′ -C-ethynylthymidine (8: 4′ -Ed4T), a recently reported promising anti-HIV agent, a new approach was developed. Since treatment of 1-(2,5-dideoxy-β-l-glycero-pent-4-enofuranosyl)thymine with Pb(OBz)4 allowed the introduction of a 4′-benzoyloxy leaving group, nucleophilic substitution at the 4′ -position became feasible for the first time. Thus, reaction between the 4′-benzoyloxy derivative (11) and Me3SiC ≡ CAl(Et)Cl as a nucleophile led to the isolation of the desired 4′-“down”-ethynyl derivative (15) stereoselectively in 62% yield.

Synthetic Use Of Epoxy-Sugar Nucleosides

Preparation of 1′,2 ′-, 3 ′,4 ′-, and 4 ′,5 ′-epoxy derivatives of nucleosides and their use for the stereoselective synthesis of 1′- and 4 ′-branched analogues are described.