Biomedical subjects

A P Grollman

Publications and source records attributed to A P Grollman.

At least 19 recordsLinked to original sources

PubMed1998

MutY DNA glycosylase: base release and intermediate complex formation.

MutY protein, a DNA glycosylase found in Escherichia coli, recognizes dA:dG, dA:8-oxodG, and dA:dC mismatches in duplex DNA, excising the adenine moiety. We have investigated the mechanism of action of MutY, addressing several points of disagreement raised by previous studies of this enzyme. MutY forms a covalent intermediate with its DNA substrate but does not catalyze strand cleavage. The covalent intermediate has a half-life of approximately 2.6 h, 2 orders of magnitude greater than the half-life of Schiff bases formed when E. coli formamidopyrimidine-DNA glycosylase (Fpg) and endonuclease III react with their respective substrates. The covalent complex between MutY and its DNA substrate involves Lys-142; however, the position of this residue in the presumptive active site differs from that of catalytic residues involved in Schiff base formation associated with endonuclease III and related DNA glycosylases/AP lyases. MutY converts DNA duplexes containing the dA:8-oxodG mispair to a product containing an abasic site; heat-induced cleavage of this product may account for the several reports in the literature that ascribe AP lyase activity to MutY. The MutY-DNA intermediate complex is highly stable and hinders access by Fpg to DNA, thereby avoiding a double-strand break. Cross-linking of MutY to DNA may play an important role in the regulation of base excision repair.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed1998

The impact of an exocyclic cytosine adduct on DNA duplex properties: significant thermodynamic consequences despite modest lesion-induced structural alterations.

The exocyclic base adduct 3,N4-deoxyethenocytosine (epsilonC) is a common DNA lesion that can arise from carcinogen exposure and/or as a biproduct of cellular processes. We have examined the thermal and thermodynamic impact of this lesion on DNA duplex properties, as well as the structural alterations imparted by the lesion. For these studies, we used calorimetric and spectroscopic techniques to investigate a family of 13-mer DNA duplexes of the form (5'CGCATGNGTACGC3')x(3'GCGTACNCATGCG5'), where the central NxN base pair represents the four standard Watson-Crick base pairs (corresponding to four control duplexes), and where either one of the N bases has been replaced by epsilonC, yielding eight test duplexes. Studies on these 12 duplexes permit us to assess the impact of the epsilonC lesion as a function of sequence context. Our spectroscopic and calorimetric data allow us to reach the following conclusions: (i) The epsilonC lesion imparts a large penalty on duplex stability, with sequence context only modestly modulating the extent of this lesion-induced destabilization. This result contrasts with our recent studies of duplexes with abasic sites, where sequence context was found to be the predominant determinant of thermodynamic damage. (ii) For the epsilonC-containing duplexes, sequence context effects are most often observed in the enthalpic contribution to lesion-induced duplex destabilization. However, due to compensating entropies, the free energy changes associated with this lesion-induced duplex destablization are nearly independent of sequence context. (iii) Despite significant lesion-induced changes in duplex energetics, our spectroscopic probes detect only modest lesion-induced changes in duplex structure. In fact, the overall duplex maintains a global B-form conformation, in agreement with NMR structural data. We discuss possible interpretations of the apparent disparity between the severe thermodynamic and relatively mild structural impacts of the epsilonC lesion on duplex properties. We also note and discuss the implications of empirical correlations between biophysical and biological properties of lesion-containing duplexes.

Base Sequence↗

PubMed1998

Mutagenic specificity of (acetylamino)fluorene-derived DNA adducts in mammalian cells.

Site-specifically modified oligodeoxynucleotides were used to explore the mutagenic potential of dG-AAF and dG-AF adducts in mammalian cells. The miscoding properties of these arylamine adducts were established by analyzing fully extended products of primer extension reactions catalyzed by mammalian DNA polymerases alpha, beta, and delta. On DNA templates containing dG-AAF, pol alpha generated two-base deletions and promoted incorporation of small amounts of dCMP, dAMP, and dTMP opposite the lesion. Reactions with pol beta were associated exclusively with two-base deletions. Primer extension catalyzed by pol delta was strongly blocked by the adduct. On DNA templates containing dG-AF, all three DNA polymerases generated full-length products, preferentially incorporating dCMP opposite the lesion. A single-stranded shuttle vector containing 5'TCCTCCTCXCCTCTC (X = dG-AAF, dG-AF, or dG) was used to establish the frequency and specificity of dG-AAF- and dG-AF-induced mutations in simian kidney (COS-7) cells. Vectors containing a single dG-AAF or dG-AF adduct promote significant incorporation of dAMP and lesser amounts of dTMP opposite the lesion. dG-AAF also promoted some incorporation of dGMP and a two-base deletion. dG-AAF was 3.8 times more mutagenic than dG-AF (11% vs 2.9%) in COS cells. We conclude from this study that dG-AAF and dG-AF produce G --> T transversions and, to a much lesser degree, G --> A transitions in mammalian cells.

2-Acetylaminofluorene↗

PubMed1998

Mutagenic potential of stereoisomeric bay region (+)- and (-)-cis-anti-benzo[a]pyrene diol epoxide-N2-2'-deoxyguanosine adducts in Escherichia coli and simian kidney cells.

We have investigated the mutagenic potential of site-specifically positioned DNA adducts with (+)- and (-)-cis-anti stereochemistry derived from the binding of r7,t8-dihydroxy-t9,10-epoxy-7,8,9, 10-tetrahydrobenzo[a]pyrene (BPDE) to N2-2'-deoxyguanosine (G1 or G2) in the sequence context 5'TCCTCCTG1 G2CCTCTC. BPDE-modified oligodeoxynucleotides were ligated to a single-stranded DNA vector and replicated in Escherichia coli or simian kidney (COS7) cells. The presence of (+)- or (-)-cis adduct strongly reduced the yield of transformants in E. coli, and the yield was improved by the induction of SOS functions. Both adducts were mutagenic in E. coli and COS cells, generating primarily G --> T transversions. In E. coli, the (-)-cis adduct was more mutagenic than the (+)-cis adduct, while in COS cells, both adducts were equally mutagenic. These results were compared with those obtained with stereoisomeric (+)- and (-)-trans adducts [Moriya, M., et al. (1996) Biochemistry 35, 16646-16651). In E. coli, cis adducts, especially (-)-cis adducts, are consistently more mutagenic than the comparable trans adduct. In COS cells, trans adducts yield higher frequencies of mutations than the two cis adducts and, with the exception of the high-mutation frequency associated with the (+)-trans adduct at G2, relatively small differences in mutation frequencies are observed for the three other adducts. In E. coli, mutation frequency is a pronounced function of adduct stereochemistry and adduct position. These findings suggest that the fidelity of translesional synthesis across BPDE-dG adducts is strongly influenced by adduct stereochemistry, nucleotide sequence context, and the DNA replication complex.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗

PubMed1998

Thermodynamic consequences of an abasic lesion in duplex DNA are strongly dependent on base sequence.

The abasic site in DNA may arise spontaneously, as a result of nucleotide base damage, or as an intermediate in glycosylase-mediated DNA-repair pathways. It is the most common damage found in DNA. We have examined the consequences of this lesion and its sequence context on DNA duplex structure, as well as the thermal and thermodynamic stability of the duplex, including the energetic origins of that stability. To this end, we incorporated a tetrahydrofuran abasic site analogue into a family of 13-mer DNA duplexes, wherein the base opposite the lesion (A, C, G, or T) and the base pairs neighboring the lesion (C.G or G.C) were systematically varied and characterized by a combination of spectroscopic and calorimetric techniques. The resulting data allowed us to reach the following conclusions: (i) the presence of the lesion in all sequence contexts studied does not alter the global B-form conformation characteristic of the parent undamaged duplex; (ii) the presence of the lesion induces a significant enthalpic destabilization of the duplex, with the magnitude of this effect being dependent on the sequence context; (iii) the thermodynamic impact of the lesion is dominated by the identity of the neighboring base pairs, with the cross strand partner base exerting only a secondary thermodynamic effect on duplex properties. In the aggregate, our data reveal that even in the absence of lesion-induced alterations in global structure, the abasic lesion can significantly alter the thermodynamic properties of the host duplex, with the magnitude of this impact being strongly dependent on sequence context.

Base Composition↗

PubMed1998

Solution structure of duplex DNA containing an extrahelical abasic site analog determined by NMR spectroscopy and molecular dynamics.

Translesional DNA synthesis past abasic sites proceeds with the preferential incorporation of dAMP opposite the lesion and, depending on the sequence context, one or two base deletions. High-resolution NMR spectroscopy and molecular dynamics simulations were used to determine the three-dimensional structure of a DNA heteroduplex containing a synthetic abasic site (tetrahydrofuran) residue positioned in a sequence that promotes one base deletions. Analysis of NMR spectra indicates that the stem region of the duplex adopts a right-handed helical structure and the glycosidic torsion angle is in anti orientation for all residues. NOE interactions establish Watson-Crick alignments for all canonical base pairs of the duplex. Measurement of distance interactions at the lesion site shows the abasic residue excluded from the helix. Restrained molecular dynamics simulations generated three-dimensional models in excellent agreement with the spectroscopic data. These structures show a regular duplex region and a slight bend at the lesion site. The tetrahydrofuran residue extrudes from the helix and is highly flexible. The model reported here, in conjunction with a previous study performed on abasic sites, explains the structural bias of one-base deletion mutations.

Computer Simulation↗

PubMed1998

Extrachromosomal unequal homologous recombination and gene conversion in simian kidney cells: effects of UV damage.

Shuttle plasmid vectors containing the SV40 origin of replication and tandem neo genes with distally placed non-overlapping deletions were used to study the effects of DNA damage on extrachromosomal homologous recombination in simian kidney cells. DNA was introduced into COS7 cells by a lipofectin-mediated transfection procedure and recombination was assessed by analyzing the structure of plasmids. Recombinational events observed included unequal homologous recombination (triplication), gene conversion, double reciprocal recombination, deletion (pop-outs), gene amplification (4-6 copies), and multimerization. Triplication, an event that previously had not been reported in association with extrachromosomal recombination, predominated in experiments with undamaged vectors. The recombination frequency (NeoR/AmpR) of vectors randomly damaged by UV irradiation was essentially unchanged; however, the relative number of triplication events decreased significantly. Selective damage in one of the two neo genes increased the relative frequency of gene conversion. The experimental system developed for use in this study detects all major homologous recombination events observed in chromosomal direct repeat sequences in mammalian cells and yeast and should prove valuable for future studies of homologous recombination in mammalian cells.

Animals↗

PubMed1997

Kinetics of DNA polymerase I (Klenow fragment exo-) activity on damaged DNA templates: effect of proximal and distal template damage on DNA synthesis.

Mutagenic DNA adducts have been analyzed with respect to the rate of nucleotide insertion opposite the modified base, extension from that "mispair", and nucleotide insertion preference. To complement and extend these studies we have investigated the long-range effects of DNA adducts on DNA polymerase activity. To address this question, primer extension reactions were performed using DNA polymerase I, Klenow fragment exo-. Templates containing 7,8-dihydro-8-oxoguanine, dG-C8-aminofluorene, dG-C8-(acetylamino)fluorene, and the model abasic site, tetrahydrofuran, were used for these studies, and the steady-state kinetics of correct nucleotide insertion were determined at positions (-2), (-1), (+1), (+2), (+3), and (+5) with respect to the template lesion. The kinetics of primer extension by Klenow fragment exo- at template positions 3' to the lesion showed only a small inhibitory effect, <3-fold, even for the strongly blocking lesion, dG-C8-(acetylamino)fluorene, indicating that Klenow fragment exo- activity is not greatly affected by lesions in the single-stranded portion of the template-primer. In contrast, a dramatic decrease in the frequency of primer extension was observed at template sites 5' to the site of adduction. Inhibition of polymerase activity decreased as the distance from the lesion increased; however, a relatively large effect was seen at the (+2) and (+3) positions for dG-C8-(acetylamino)fluorene and tetrahydrofuran. For these blocking lesions, the effect on extension 5 bases from the lesion was greatly reduced. We conclude from these studies that DNA damage at positions remote from the site of the lesion affects DNA polymerase function.

DNA Damage↗

PubMed1997

Solution structure of a DNA duplex containing the exocyclic lesion 3,N4-etheno-2'-deoxycytidine opposite 2'-deoxyguanosine.

Vinyl chloride reacts with cellular DNA producing 3,N4-etheno-2'-deoxycytidine (epsilonC) along with other exocyclic adducts. The solution structure of an oligodeoxynucleotide duplex containing an epsilonC.dG base pair was determined by high-resolution NMR spectroscopy and molecular dynamics simulations. NMR data indicated that the duplex adopts a right-handed helical structure having all residues in anti orientation around the glycosylic torsion angle. The epsilonC adduct has a sugar pucker in the C3'-endo/C4'-exo region while the rest of the residues are in the C2'-endo/C3'-exo range. NOE interactions established Watson-Crick alignments for canonical base pairs of the duplex. The imino proton of the lesion-containing base pair resonated as a sharp signal that was resistant to water exchange, suggesting hydrogen bonding. Restrained molecular dynamics simulations generated three-dimensional models in excellent agreement with the spectroscopic data. The refined structures are slightly bent at the lesion site without major perturbations of the sugar-phosphate backbone. The adduct is displaced and shifted toward the major groove of the helix while its partner on the complementary strand remains stacked. The epsilonC(anti).dG(anti) base pair alignment is sheared and stabilized by the formation of hydrogen bonds. The biological implications of structures of epsilonC-containing DNA duplexes are discussed.

DNA↗

PubMed1997

Cloning and characterization of a mammalian 8-oxoguanine DNA glycosylase.

Oxidative DNA damage is generated by reactive oxygen species. The mutagenic base, 8-oxoguanine, formed by this process, is removed from oxidatively damaged DNA by base excision repair. Genes coding for DNA repair enzymes that recognize 8-oxoguanine have been reported in bacteria and yeast. We have identified and characterized mouse and human cDNAs encoding homologs of the 8-oxoguanine DNA glycosylase (ogg1) gene of Saccharomyces cerevisiae. Escherichia coli doubly mutant for mutM and mutY have a mutator phenotype and are deficient in 8-oxoguanine repair. The recombinant mouse gene (mOgg1) suppresses the mutator phenotype of mutY/mutM E. coli. Extracts prepared from mutY/mutM E. coli expressing mOgg1 contain an activity that excises 8-oxoguanine from DNA and a beta-lyase activity that nicks DNA 3' to the lesion. The mouse ogg1 gene product acts efficiently on DNA duplexes in which 7, 8-dihydroxy-8-oxo-2'-deoxyguanosine (8-oxodG) is paired with dC, acts weakly on duplexes in which 8-oxodG is paired with dT or dG, and is inactive against duplexes in which 8-oxodG is paired with dA. Mouse and human ogg1 genes contain a helix-hairpin-helix structural motif with conserved residues characteristic of a recently defined family of DNA glycosylases. Ogg1 mRNA is expressed in several mouse tissues; highest levels were detected in testes. Isolation of the mouse ogg1 gene makes it possible to modulate its expression in mice and to explore the involvement of oxidative DNA damage and associated repair processes in aging and cancer.

Amino Acid Sequence↗

PubMed1997

Translesional synthesis on DNA templates containing a single abasic site. A mechanistic study of the "A rule".

Site-specifically modified oligodeoxynucleotides containing a single natural abasic site or a chemically synthesized (tetrahydrofuran or deoxyribitol) model abasic site were used as templates for primer extension reactions catalyzed by the Klenow fragment of Escherichia coli DNA polymerase I or by calf thymus DNA polymerase alpha. Analysis of the fully extended products of these reactions indicated that both polymerases preferentially incorporate dAMP opposite the natural abasic site and tetrahydrofuran, while DNA templates containing the ring-opened deoxyribitol moiety block translesional synthesis, promoting sequence context-dependent deletions. The frequency of nucleotide insertion opposite the three types of abasic sites follows the order dAMP > dGMP > dCMP > dTMP. The frequency of chain extension was highest when dAMP was positioned opposite a natural abasic site. The frequency of translesional synthesis past abasic sites follows the order tetrahydrofuran > deoxyribose > deoxyribitol. The Klenow fragment promotes blunt end addition of dAMP; this reaction was much less efficient than insertion of dAMP opposite an abasic site. We conclude that the miscoding potential of a natural abasic site in vitro closely resembles that of its tetrahydrofuran analog. Ring-opened abasic sites favor deletions. Studies with polymerase alpha in vitro predict preferential incorporation of dAMP at abasic sites in mammalian cells.

Animals↗

PubMed1997

Molecular mechanisms of mutagenesis by aromatic amines and amides.

The mutagenic properties of 2-acetylaminofluorene-derived DNA adducts, including N-(deoxyguanosin-8-yl)-2-acetylaminofluorene, N-(deoxyguanosin-8-yl)-2-aminofluorene, N-(deoxyguanosin-N2-yl)-2-acetylaminofluorene, and several minor oxidation products have been explored, using site-specific techniques. Oligodeoxynucleotides containing a single AAF-derived DNA adduct were prepared by postsynthetic modification and used as templates in primer extension reactions catalyzed by bacterial and mammalian DNA polymerases. Base substitutions and deletions occurring during DNA synthesis were quantified. dG-C8-AAF promoted one- and two-base deletions and small amounts of incorporation of dCMP, dAMP, and/or dTMP opposite the lesion in reactions catalyzed by the 3'-->5' exonuclease-free Klenow fragment of DNA polymerase 1 (exo-) and polymerase alpha. dG-C8-AF did not miscode in reactions catalyzed by exo-; however, base misincorporation and deletions were observed in reactions with pol alpha. dG-N2-AAF promoted small amounts of dAMP incorporation in reactions catalyzed by exo-. The miscoding potential of minor oxidation products of dG-C8-AF was much higher than that of other adducts. Steady-state kinetics were used to measure frequencies of nucleotide insertion opposite the lesion and chain extension from the 3' terminus. Kinetic data were consistent with the results of primer extension studies. A mutation 'hot spot' was constructed and the influence of sequence context on the frequency of deletions generated by dG-C8-AAF was explored systematically in reactions catalyzed by exo-. Based on our results with aminofluorene DNA adducts, we propose a general mechanism for frameshift deletion mutagenesis. Site-specific methods also were used to establish the mutagenic potential of AAF-derived DNA adducts in mammalian cells. dG-C8-AAF and dG-C8-AF exhibited similar mutagenic specificities, predicting the occurrence of G-->T transversions and G-->A transitions in mammalian cells.

2-Acetylaminofluorene↗

PubMed1997

NH2-terminal proline acts as a nucleophile in the glycosylase/AP-lyase reaction catalyzed by Escherichia coli formamidopyrimidine-DNA glycosylase (Fpg) protein.

Formamidopyrimidine-DNA glycosylase (Fpg) protein plays a prominent role in the repair of oxidatively damaged DNA in Escherichia coli. The protein possesses three enzymatic activities, hydrolysis of the N-glycosidic bond (DNA glycosylase), beta-elimination (AP lyase), and delta-elimination; these functions act in a concerted manner to excise oxidized deoxynucleosides from duplex DNA. Schiff base formation between the enzyme and substrate has been demonstrated (Tchou, J., and Grollman, A. P. (1995) J. Biol. Chem. 270, 11671-11677); this protein-DNA complex can be trapped by reduction with sodium borohydride. By digesting the stable, covalently linked intermediate with proteases and determining the accurate mass of the products by negative electrospray ionization-mass spectrometry, we show that the N-terminal proline of Fpg protein is linked to DNA and, therefore, is identified as the nucleophile that initiates the catalytic excision of oxidized bases from DNA. This experimental approach may be applicable to the analysis of other protein-DNA complexes.

Amino Acid Sequence↗

PubMed1997

Kinetics of excision of purine lesions from DNA by Escherichia coli Fpg protein.

The kinetics of excision of damaged purine bases from oxidatively damaged DNA by Escherichia coli Fpg protein were investigated. DNA substrates, prepared by treatment with H2O2/Fe(III)-EDTA or by gamma-irradiation under N2O or air, were incubated with Fpg protein, followed by precipitation of DNA. Precipitated DNA and supernatant fractions were analyzed by gas chromatography/isotope-dilution mass spectrometry. Kinetic studies revealed efficient excision of 8-hydroxyguanine (8-OH-Gua), 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) and 4, 6-diamino-5-formamidopyrimidine (FapyAde). Thirteen other modified bases in the oxidized DNA substrates, including 5-hydroxycytosine and 5-hydroxyuracil, were not excised. Excision was measured as a function of enzyme concentration, substrate concentration, time and temperature. The rate of release of modified purine bases from the three damaged DNA substrates varied significantly even though each DNA substrate contained similar levels of oxidative damage. Specificity constants (kcat/KM) for the excision reaction indicated similar preferences of Fpg protein for excision of 8-OH-Gua, FapyGua and FapyAde from each DNA substrate. These findings suggest that, in addition to 8-OH-Gua, FapyGua and FapyAde may be primary substrates for this enzyme in cells.

Animals↗

PubMed1996

Fidelity of translesional synthesis past benzo[a]pyrene diol epoxide-2'-deoxyguanosine DNA adducts: marked effects of host cell, sequence context, and chirality.

We have used a site-specific approach to investigate the mutagenic potential of (+)- and (-)-trans-anti-benzo[a]pyrene diol epoxide (BPDE) DNA adducts. Oligodeoxyribonucleotides (5'TCCTCCTG1G2-CCTCTC), modified at the exocyclic amino groups of G1 or G2, were incorporated into a single-stranded shuttle vector and introduced into Escherichia coli or simian kidney (COS) cells. This experimental system permits translesional synthesis to proceed in the absence of DNA repair. The presence of (+)- or (-)-BPDE-N2-dG adducts strongly inhibited translesional synthesis in E. coli; induction of cellular SOS functions reduced this blocking effect. Vectors containing (+)-BPDE adducts at G1 or G2 generated mutation frequencies of 19% and 3%, respectively; these values were not altered significantly by induction of SOS functions. In COS cells, (+)-BPDE-modified vectors generated mutation frequencies of 13% at G1 and 45% at G2. In E. coli, the (-)-BPDE adduct generated mutation frequencies of < or = 2% at G1 and G2 and, in COS cells, 13% at G1 and 21% at G2. The predominant mutations in E. coli and COS cells were G-->T transversions targeted to the site of the lesion; however, when G2 was modified, a significant number of targeted G-->A and G-->C mutations were observed in COS cells. We conclude from this study that (+)-and (-)-BPDE-N2-dG adducts pair preferentially to dCMP and dAMP during translesional synthesis in a process that is strongly influenced by the stereochemistry of the adduct, by the bases flanking the lesion, and by host cell factors.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗

PubMed1996

Miscoding properties of 3,N4-etheno-2'-deoxycytidine in reactions catalyzed by mammalian DNA polymerases.

We have investigated the miscoding properties of the exocyclic DNA adduct, 3,N4-etheno-2'-deoxycytidine (epsilon dC), using an experimental system designed to detect and quantify base substitutions and deletions generated by primer extension in reactions catalyzed by DNA polymerases alpha, beta, and delta. Oligodeoxynucleotides modified site-specifically with epsilon dC were used as DNA templates for this study. Pol alpha catalyzed incorporation of dTMP and dAMP opposite epsilon dC, accompanied by lesser amounts of dCMP and dGMP and some two-base deletions. Pol beta promoted incorporation of dCMP and dAMP, along with small amounts of one-base and two-base deletions. Pol delta catalyzed incorporation of dTMP and lesser amounts of dAMP and dGMP. The frequency of nucleotide insertion opposite epsilon dC and of chain extension from the 3'-primer terminus in reactions catalyzed by pol alpha and pol beta was established by steady-state kinetic analysis. Results of this study were consistent with those obtained in primer extension experiments. The miscoding properties of epsilon dC determined in vitro are consistent with observations of epsilon dC-->A transversions and epsilon dC-->T transitions in site-specific mutagenesis experiments in mammalian cells (Moriya et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 11899). We conclude from this study that DNA polymerases may differ significantly in their miscoding potential and that in vitro analysis can be used to predict mutagenic specificity of exocyclic DNA adducts in mammalian cells.

Animals↗

PubMed1996

Solution structure of an oligodeoxynucleotide duplex containing the exocyclic lesion 3,N4-etheno-2'-deoxycytidine opposite 2'-deoxyadenosine, determined by NMR spectroscopy and restrained molecular dynamics.

The d(C-G-T-A-C-epsilon C-C-A-T-G-C).d(G-C-A-T-G-A-G-T-A-C-G) oligodeoxynucleotide duplex containing the 3, N4-etheno-2'-deoxycytidine adduct positioned opposite 2'-deoxyadenosine in the center of the helix has been analyzed by proton NMR spectroscopy and restrained molecular dynamics. The spectroscopic data establish a right-handed duplex, with sugar puckers in the C2'-endo/C3'-exo range, residues adopting an anti conformation around the glycosidic torsion angle and, with the exception of epsilon C.dA, Watson-Crick hydrogen bond alignment for all base pairs. Molecular dynamics simulations, restrained by the full relaxation matrix approach, produced a three-dimensional model with an NMR R-factor of 7%. The duplex structure shows no significant perturbation of the sugar-phosphate backbone, which remains in B-form. The exocyclic adduct and its partner dA are incorporated into the helix without producing a noticeable kink. The epsilon C.dA alignment adopts a staggered conformation with each residue displaced toward the 5'-terminus and intercalated between bases on the opposite strand, without increase of inter-phosphate distances. The partial intercalation of the epsilon C (anti).dA(anti) alignment allows stacking between the aromatic rings of epsilon C and dA and with base pairs adjacent to the lesion, suggesting an important role played by hydrophobic forces in the stabilization of the solution structure.

Base Composition↗

PubMed1996

NMR solution structure of an oligodeoxynucleotide duplex containing the exocyclic lesion 3,N4-etheno-2'-deoxycytidine opposite thymidine: comparison with the duplex containing deoxyadenosine opposite the adduct.

The exocyclic 3,N4-etheno-2'-deoxycytidine adduct was incorporated at the center of the oligodeoxynucleotide duplex d(C-G-T-A-C-epsilon C-C-A-T-G-C).d (G-C-A-T-G-T-G-T-A-C-G), and its solution structure was analyzed using high-resolution proton NMR spectroscopy and molecular dynamics simulations. The experimental data indicate that the oligodeoxynucleotide duplex adopts a right-handed helical structure with sugar puckers in the C2'-endo/C3'-exo range and Watson-Crick hydrogen bond alignments for all base pairs. NOE connectivities established a syn orientation for the glycosidic torsion angle of the exocyclic adduct. Restrained molecular dynamics simulations, using the full relaxation matrix approach, produced a three-dimensional model in agreement with the experimental data. The structure shows only minor perturbations in the sugar-phosphate backbone and a 27 degrees bend of the helical axis at the lesion site. On the refined model a well-formed hydrogen bond between T (N3H) and epsilon C(N4) stabilizes the epsilon C(syn).T(anti) base pair alignment, reflecting the preference of the adduct for the syn orientation. Furthermore, the epsilon C(syn).T(anti) base pair stacks with flanking base pairs. We discuss a correlation between the mutagenic properties of the adduct and the three-dimensional structure of the epsilon C.dA and epsilon C.T duplexes.

Base Composition↗