The Mutagenicity of Thymidine Glycol in <i>Escherichia coli</i> Is Increased When It Is Part of a Tandem Lesion

Tandem lesions are comprised of two contiguously damaged nucleotides. Tandem lesions make up the major family of reaction products generated from a pyrimidine nucleobase radical, which are formed in large amounts by ionizing radiation. One of these tandem lesions contains a thymidine glycol lesion flanked on its 5′-side by 2-deoxyribonolactone (LTg). The replication of this tandem lesion was investigated in Escherichia coli using single-stranded genomes. LTg is a much more potent replication block than thymidine glycol and is bypassed only under SOS-induced conditions. The adjacent thymidine glycol does not significantly affect nucleotide incorporation opposite 2-deoxyribonolactone in wild-type cells. In contrast, the misinsertion frequency opposite thymidine glycol, which is negligible in the absence of 2-deoxyribonolactone, increases to 10% in wild-type cells when LTg is flanked by a 3′-dG. Experiments in which the flanking nucleotides are varied and in cells lacking one of the SOS-induced bypass polymerases indicate that the mutations are due to a mechanism in which the primer misaligns prior to bypassing the lesion, which allows for an additional nucleotide to be incorporated across from the 3′-flanking nucleotide. Subsequent realignment and extension results in the observed mutations. DNA polymerases II and IV are responsible for misalignment induced mutations and compete with DNA polymerase V which reads through the tandem lesion. These experiments reveal that incorporation of the thymidine glycol into a tandem lesion indirectly induces increases in mutations by blocking replication, which enables the misalignment−realignment mechanism to compete with direct bypass by DNA polymerase V

Synthesis, DNA Polymerase Incorporation, and Enzymatic Phosphate Hydrolysis of Formamidopyrimidine Nucleoside Triphosphates

The nucleoside triphosphates of N6-(2-deoxy-α,β-d-erythro-pentofuranosyl)-2,6-diamino-4-hydroxy-5-formamidopyrimidine (Fapy·dGTP) and its C-nucleoside analogue (β-C-Fapy·dGTP) were synthesized. The lability of the formamide group required that nucleoside triphosphate formation be carried out using an umpolung strategy in which pyrophosphate was activated toward nucleophilic attack. The Klenow fragment of DNA polymerase I from Escherichia coli accepted Fapy·dGTP and β-C-Fapy·dGTP as substrates much less efficiently than it did dGTP. Subsequent extension of a primer containing either modified nucleotide was less affected compared to when the native nucleotide is present at the 3‘-terminus. The specificity constants are sufficiently large that nucleoside triphosphate incorporation could account for the level of Fapy·dG observed in cells if 1% of the dGTP pool is converted to Fapy·dGTP. Similarly, polymerase-mediated introduction of β-C-Fapy·dG could be useful for incorporating useful amounts of this nonhydrolyzable analogue for use as an inhibitor of base excision repair. The kinetic viability of these processes is enhanced by inefficient hydrolysis of Fapy·dGTP and β-C-Fapy·dGTP by MutT, the E. coli enzyme that releases pyrophosphate and the corresponding nucleoside monophosphate upon reaction with structurally related nucleoside triphosphates

DNA Tandem Lesion Repair by Strand Displacement Synthesis and Nucleotide Excision Repair

DNA tandem lesions are comprised of two contiguously damaged nucleotides. This subset of clustered lesions is produced by a variety of oxidizing agents, including ionizing radiation. Clustered lesions can inhibit base excision repair (BER). We report the effects of tandem lesions composed of a thymine glycol and a 5′-adjacent 2-deoxyribonolactone (LTg) or tetrahydrofuran abasic site (FTg). Some BER enzymes that act on the respective isolated lesions do not accept the tandem lesion as a substrate. For instance, endonuclease III (Nth) does not excise thymine glycol (Tg) when it is part of either tandem lesion. Similarly, endonuclease IV (Nfo) does not incise L or F when they are in tandem with Tg. Long-patch BER overcomes inhibition by the tandem lesion. DNA polymerase β (Pol β) carries out strand displacement synthesis, following APE1 incision of the abasic site. Pol β activity is enhanced by flap endonuclease (FEN1), which cleaves the resulting flap. The tandem lesion is also incised by the bacterial nucleotide excision repair system UvrABC with almost the same efficiency as an isolated Tg. These data reveal two solutions that DNA repair systems can use to counteract the formation of tandem lesions

Pharmacokinetic properties of a novel inosine analog, 4′-cyano-2′-deoxyinosine, after oral administration in rats

<div><p>4′-cyano-2′-deoxyinosine (SK14-061a), a novel nucleoside analog based on inosine, has antiviral activity against the human immunodeficiency virus type 1 that has the ability to acquire resistance against many types of reverse transcriptase inhibitors based on nucleosides. The aim of this study was to investigate the pharmacokinetics studies after its oral administration to rats. For this purpose, we first developed and validated an analytical method for quantitatively determining SK14-061a levels in biological samples by a UPLC system interfaced with a TOF-MS system. A rapid, simple and selective method for the quantification of SK14-061a in biological samples was established using liquid chromatography mass spectrometry (LC-MS) with solid phase extraction. The pharmacokinetic properties of SK14-061a in rats after oral administration were then evaluated using this LC-MS method. SK14-061a was found to be relatively highly bioavailable, is rapidly absorbed from the intestinal tract, and is then mainly distributed to the liver and then ultimately excreted via the urine in an unchanged form. Furthermore, the simultaneous administration of SK14-061a with the nucleoside analog, entecavir, led to a significant alteration in the pharmacokinetics of SK14-061a. These results suggest that the SK14-061a has favorable pharmacokinetic properties with a high bioavailability with the potential for use in oral pharmaceutical formulations, but drug-drug interactions should also be considered.</p></div

Time course for the plasma concentration of SK14-061a after oral administration at a dose of 1 mg/kg in rats.

Venous blood samples were collected at 15 min, 45 min, 90 min, 3, 4.5, 6 and 9 hr after oral administration. The SK14-061a concentrations in plasma were measured by LC-MS combined with the SPE method. The values are the mean ± SD. (n = 4).</p

Intra- and inter-day accuracy and precision for SK14-061a in plasma.

<p>Intra- and inter-day accuracy and precision for SK14-061a in plasma.</p

LC-MS chromatograms of (A) SK14-061a spiked with water, (B) SK14-061a spiked with plasma after a methanol treatment, (C) SK14-061a spiked with plasma after an acetonitrile treatment, (D) blank plasma after SPE treatment, and (E) SK14-061a spiked with plasma after SPE treatment.

<p>All SK14-061a samples contained 500 ng/mL in concentration before either reverse liquid-liquid extraction or SPE. The SK14-061a were detected by the LC-MS as described in the Materials and Methods section.</p

Pharmacokinetic parameters of SK14-061a after intravenous and oral administration of a dose of 1 mg/kg in rats.

<p>Pharmacokinetic parameters of SK14-061a after intravenous and oral administration of a dose of 1 mg/kg in rats.</p

Pharmacokinetic parameters of SK14-061a and ETV after the oral co-administration of SK14-061a and ETV at a dose of 1 mg/kg in rats.

<p>Pharmacokinetic parameters of SK14-061a and ETV after the oral co-administration of SK14-061a and ETV at a dose of 1 mg/kg in rats.</p

LC-MS chromatograms of SK14-061a in urine.

<p>Urine samples were collected in a metabolic cage and were pretreated on an SPE column (Waters Oasis MCX 96 well plate) for extracting SK14-061a as described in the Materials and Methods section.</p