Search

The conformations of C8-dG adducts of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) in the recognition sequence of the NarI restriction enzyme are compared. The sequence-dependent differences have indicated that base-displaced intercalation of the IQ adduct is favored when both the 5'- and 3'-flanking nucleotides in the complementary strand are guanines, thus correlating with sequence-dependent differences in translesion replication.

The solution structure of the oligodeoxynucleotide 5'-d(CTCGGCXCCATC)-3',5'-d(GATGGCGCCGAG)-3' containing the heterocyclic amine 8-[(3-methyl-3H-imidazo[4,5-f]quinolin-2-yl)amino]-2'-deoxyguanosine adduct (IQ) at the third guanine in the Narl restriction sequence, a hot spot for -2 bp frameshifts, is presented. The results have confirmed that IQ has adopted a base-displaced intercalated conformation in this sequence context but have not supported the formation of a hydrogen bond between the IQ quinoline nitrogen and the complementary dC.

An efficient and general strategy for the synthesis of C8-deoxyguanosine adducts of arylamines is developed, which involve the Buchwald-Hartwig palladium-catalyzed N-arylation reaction of a suitably protected 8-bromo-2'-deoxyguanosine derivative 16 with various mutagenic arylamines including the dietary mutagen. Phosphoramidite approach is used to site-specifically incorporate the C8-IQ-adducted nucleoside into oligonucleotides.

Deamination of nucleobases in DNA and RNA results in the formation of xanthine (X), hypoxanthine (I), oxanine, and uracil, all of which are miscoding and mutagenic in DNA and can interfere with RNA editing and function. Among many forms of nucleic acid damage, deamination arises from several unrelated mechanisms, including hydrolysis. nitrosative chemistry, and deaminase enzymes. Here we present a fourth mechanism contributing to th~ burden of nucleobase deamination: incorporation of hypoxanthine and xanthine into DNA and RNA caused by defects in purine nucleotide metabolism. Using Escherichia coil and Saccharomyces cerevisiae with defined mutations in purine metabolism in conjunction with analytical methods for quantifying deaminated nucleobases in DNA and RNA. we observed large increases (up to 600-fold) in hypoxanthine in both DNA and RNA in cells unable to convert IMP to XMP or AMP (IMP dehydrogenase, guaB; adenylosuccinate synthetase, purA. and ADE12), and unable to remove dITP/ITP and dXTP/XTP from the nucleotide pool (dITP/XTP pyrophosphohydrolase, rdgB and HAMI). Conversely, modest changes in xanthine levels were observed in RNA (but not DNA) from E. co/i lacking purA and rdgB and the enzyme converting XMP to GMP (GMP synthetase, guaA). These observations suggest that disturbances in purine metabolism caused by known genetic polymorphisms could increase the burden of mutagenic deaminated nucleobases in DNA and interfere with gene expression and RNA function, a situation possibly exacerbated by the nitrosative stress of concurrent inflammation. The results also suggest a mechanistic basis for the pathophysiology of human inborn errors of purine nucleotide metabolism. [ABSTRACT FROM AUTHOR]

The conformations of C8-dG adducts of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) positioned in the C-X¹-G, G-X²-C, and C-X³-C contexts in the C-G¹-G²-C-G³-C-C recognition sequence of the Nan restriction enzyme were compared, using the oligodeoxynucleotides 5′-d(CTCXGCGCCATC)-3′5′-d(GATGGCGCCGAG)-3′, 5′-d(CTCGXCGCCATC)-3′5′-d(GATGGCGCCGAG)-3′, and 5′- d(CTCGGCXCCATC)-3′·5′-d(GATGGCGCCGAG)-3′ (X is the C8-dG adduct of IQ). These were the NarIIQ1, NarIIQ2, and NarIIQ3 duplexes, respectively. In each instance, the glycosyl torsion angle x for the IQ-modified dG was in the syn conformation. The orientations of the IQ moieties were dependent upon the conformations of torsion angles α′ [N9-C8-N(IQ)-C2(IQ)] and β′ [C8-N(IQ)-C2(IQ)-N3(IQ)I, which were monitored by the patterns of ¹H NOEs between the IQ moieties and the DNA in the three sequence contexts. The conformational states of IQ torsion angles β′ and β′ were predicted from the refined structures of the three adducts obtained from restrained molecular dynamics calculations, utilizing simulated annealing protocols. For the NarIIQ1 and NarIIQ2 duplexes, the β′ torsion angles were predicted to be -176 ± 8° and -160 ± 8°, respectively, whereas for the NarIIQ3 duplex, torsion angle β′ was predicted to be 159 ± 7°. Likewise, for the NarIIQ1 and NarIIQ2 duplexes, the β′ torsion angles were predicted to be -152 + 8° and -164 ± 7°, respectively, whereas for the NarIIQ3 duplex, torsion angle β′ was predicted to be -23 ± 8°. Consequently, the conformations of the IQ adduct in the NarIIQ1 and NarIIQ2 duplexes were similar, with the IQ methyl protons and IQ H4 and H5 protons facing outward in the minor groove, whereas in the NarIIQ3 duplex, the IQ methyl protons and the IQ H4 and H5 protons faced into the DNA duplex, facilitating the base-displaced intercalated orientation of the IQ moiety [Wang, F., Elmquist, C. E., Stover, J. S., Rizzo, C. J., and Stone, M. P. (2006) J. Am. Chem. Soc. 128, 10085- 10095]. In contrast, for the NarIIQ1 and NarIIQ2 duplexes, the IQ moiety remained in the minor groove. These sequence-dependent differences suggest that base-displaced intercalation of the IQ adduct is favored when both the 5′- and 3′-flanking nucleotides in the complementary strand are guanines. These conformational differences may correlate with sequence-dependent differences in translesion replication. [ABSTRACT FROM AUTHOR]

The site-specific synthesis of oligonucleotides containing the C8-deoxyguanosine adduct of the highly mutagenic heterocyclic amine 2-amino-3-methylimidazo[4,5-flquinoline (IQ) has been achieved, and the oligonucleotides were characterized by UV melting temperature analysis, circular dichroism, and UV absorption spectroscopy. Examination of these data indicated that the IQ-adduct is accommodated in dramatically different environments. This sequence-dependent conformational preference is likely to play a key role in the mutagenicity and repair of IQ-modified oligonucleotides. [ABSTRACT FROM AUTHOR]

The solution structure of the oligodeoxynucleotide 5'-d(CTCGGCXCCATC)-3'·5'-d(GATGGCGC-CGAG)-3' containing the heterocyclic amine 8-[(3-methyl-3H-imidazo[4,5-l]quinolin-2-yl)amino]-2'-deoxyguanosine adduct (IQ) at the third guanine in the Narl restriction sequence, a hot spot for -2 bp frameshifts, is reported. Molecular dynamics calculations restrained by distances derived from 24 ¹H NOEs between IQ and DNA, and torsion angles derived from ³J couplings, yielded ensembles of structures in which the adducted guanine was displaced into the major groove with its glycosyl torsion angle in the syn conformation. One proton of its exocyclic amine was approximately 2.8 Å from an oxygen of the 5' phosphodiester linkage, suggesting formation of a hydrogen bond. The carcinogen-guanine linkage was defined by torsion angles α' [N9-C8-N(IQ)-C2(IQ)] of 159 ± 7° and β' [C8-N(IQ)-C2(IQ)-N3(IQ)J of -23 ± 8°. The complementary cytosine was also displaced into the major groove. This allowed IQ to intercalate between the flanking C·G base pairs. The disruption of Watson–Crick hydrogen bonding was corroborated by chemical-shift perturbations for base aromatic protons in the complementary strand opposite to the modified guanine. Chemical-shift perturbations were also observed for 31P resonances corresponding to phosphodiester linkages flanking the adduct. The results confirmed that IQ adopted a base-displaced intercalated conformation in this sequence context but did not corroborate the formation of a hydrogen bond between the IQ quinoline nitrogen and the complementary dC [Elmquist, C. E.; Stover, J. S.; Wang, Z.; Rizzo, C. J. J. Am. Chem. Soc. 2004, 126, 11189-11201]. [ABSTRACT FROM AUTHOR]

Nitric oxide (NO) plays key roles in cell signaling and physiology, with diverse functions mediated by NO concentrations varying over three orders-of-magnitude. In spite of this critical concentration dependence, current approaches to NO delivery in vitro result in biologically irrelevant and poorly controlled levels, with hyperoxic conditions imposed by ambient air. To solve these problems, we developed a system for controlled delivery of NO and O(2) over large concentration ranges to mimic biological conditions. Here we describe the fabrication, operation and calibration of the delivery system. We then describe applications for delivery of NO and O(2) into cell culture media, with a comparison of experimental results and predictions from mass transfer models that predict the steady-state levels of various NO-derived reactive species. We also determined that components of culture media do not affect the steady-state levels of NO or O(2) in the device. This system provides critical control of NO delivery for in vitro models of NO biology and chemistry., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The solution structure of the oligodeoxynucleotide 5'-d(CTCGGCXCCATC)-3'.5'-d(GATGGCGCCGAG)-3' containing the heterocyclic amine 8-[(3-methyl-3H-imidazo[4,5-f]quinolin-2-yl)amino]-2'-deoxyguanosine adduct (IQ) at the third guanine in the NarI restriction sequence, a hot spot for -2 bp frameshifts, is reported. Molecular dynamics calculations restrained by distances derived from 24 (1)H NOEs between IQ and DNA, and torsion angles derived from (3)J couplings, yielded ensembles of structures in which the adducted guanine was displaced into the major groove with its glycosyl torsion angle in the syn conformation. One proton of its exocyclic amine was approximately 2.8 A from an oxygen of the 5' phosphodiester linkage, suggesting formation of a hydrogen bond. The carcinogen-guanine linkage was defined by torsion angles alpha' [N9-C8-N(IQ)-C2(IQ)] of 159 +/- 7 degrees and beta' [C8-N(IQ)-C2(IQ)-N3(IQ)] of -23 +/- 8 degrees . The complementary cytosine was also displaced into the major groove. This allowed IQ to intercalate between the flanking C.G base pairs. The disruption of Watson-Crick hydrogen bonding was corroborated by chemical-shift perturbations for base aromatic protons in the complementary strand opposite to the modified guanine. Chemical-shift perturbations were also observed for (31)P resonances corresponding to phosphodiester linkages flanking the adduct. The results confirmed that IQ adopted a base-displaced intercalated conformation in this sequence context but did not corroborate the formation of a hydrogen bond between the IQ quinoline nitrogen and the complementary dC [Elmquist, C. E.; Stover, J. S.; Wang, Z.; Rizzo, C. J. J. Am. Chem. Soc. 2004, 126, 11189-11201].