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NMR Characterization of a DNA Duplex Containing the Major Acrolein-derived Deoxyguanosine Adduct γ-OH-1,-N 2-Propano-2′-deoxyguanosine

2001; Elsevier BV; Volume: 276; Issue: 12 Linguagem: Inglês

10.1074/jbc.m009028200

ISSN

1083-351X

Autores

Carlos de los Santos, Tanya Zaliznyak, Francis Johnson,

Tópico(s)

RNA Interference and Gene Delivery

Resumo

The environmental and endogenous mutagen acrolein reacts with cellular DNA to produce several isomeric 1,N 2-propanodeoxyguanosine adducts. High resolution NMR spectroscopy was used to establish the structural features of the major acrolein-derived adduct, γ-OH-1,N 2-propano-2′-deoxyguanosine. In aqueous solution, this adduct was shown to assume a ring-closed form. In contrast, when γ-OH-1,N 2-propano-2′-deoxyguanosine pairs with dC at the center of an 11-mer oligodeoxynucleotide duplex, the exocyclic ring opens, enabling the modified base to participate in a standard Watson-Crick base pairing alignment. Analysis of the duplex spectra reveals a regular right-handed helical structure with all residues adopting an anti orientation around the glycosidic torsion angle and Watson-Crick alignments for all base pairs. We conclude from this study that formation of duplex DNA triggers the hydrolytic conversion of γ-OH-1,N 2-propano-2′-deoxyguanosine to an open chain form, a structure that facilitates pairing with dC during DNA replication and accounts for the surprising lack of mutagenicity associated with this DNA adduct. The environmental and endogenous mutagen acrolein reacts with cellular DNA to produce several isomeric 1,N 2-propanodeoxyguanosine adducts. High resolution NMR spectroscopy was used to establish the structural features of the major acrolein-derived adduct, γ-OH-1,N 2-propano-2′-deoxyguanosine. In aqueous solution, this adduct was shown to assume a ring-closed form. In contrast, when γ-OH-1,N 2-propano-2′-deoxyguanosine pairs with dC at the center of an 11-mer oligodeoxynucleotide duplex, the exocyclic ring opens, enabling the modified base to participate in a standard Watson-Crick base pairing alignment. Analysis of the duplex spectra reveals a regular right-handed helical structure with all residues adopting an anti orientation around the glycosidic torsion angle and Watson-Crick alignments for all base pairs. We conclude from this study that formation of duplex DNA triggers the hydrolytic conversion of γ-OH-1,N 2-propano-2′-deoxyguanosine to an open chain form, a structure that facilitates pairing with dC during DNA replication and accounts for the surprising lack of mutagenicity associated with this DNA adduct. γ-OH-1,N 2-propano-2′-deoxyguanosine (2,2,3,3-d4)sodium 3-trimethylsilyl-propionate nuclear Overhauser effect spectroscopy correlation spectroscopy total correlation spectroscopy nuclear Overhauser effect 2′-deoxycytidine 2′-deoxyadenosine 2′-deoxyguanosine thymidine acrolein Acrolein is a ubiquitous environmental pollutant formed by incomplete combustion of organic materials, including wood, food, tobacco, and fuels. This α,β-unsaturated aldehyde reacts to form hydroxylated 1,N 2-propano-2′-deoxyguanosine adducts in DNA (1World Health Organization Publications (1992) The WHO Environmental Health Criteria Series, Vol. 127.Google Scholar, 2Galliani G. Pantarotto C. Tetrahedron Lett. 1983; 24: 4491-4492Crossref Scopus (29) Google Scholar, 3Chung F.L. Young R. Hecht S.S. Cancer Res. 1984; 44: 990-995PubMed Google Scholar). Acrolein also is formed endogenously during the metabolic oxidation of polyamines (4Lee Y. Sayre L.M. J. Biol. Chem. 1998; 273: 19490-19494Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar) and is an end product of lipid peroxidation (5Esterbauer H. Schaur R.J. Zoller H. Free Radic. Biol. Med. 1991; 11: 81-128Crossref PubMed Scopus (6032) Google Scholar, 6Wu H.-Y. Lin Y.-L. Anal. Chem. 1995; 76: 1603-1612Crossref Scopus (34) Google Scholar, 7Chung F.L. Nath R.G. Nagao M. Nishikawa A. Zhou G.D. Randerath K. Mutat. Res. 1999; 242: 71-81Crossref Scopus (143) Google Scholar). γ-OH-1,N 2-propano-2′-deoxyguanosine (γ-OH-PdG)1 adducts were detected in DNA extracted from rat and human liver (8Nath R.G. Chung F.-L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7491-7495Crossref PubMed Scopus (222) Google Scholar, 9Nath R.G. Ocando J.E. Chung F.-L. Cancer Res. 1996; 56: 452-456PubMed Google Scholar, 10Chung F.-L. Zhang L. Ocando J.E. Nath R.G. IARC Sci. Publ. 1999; 150: 45-54PubMed Google Scholar) and from lymphocyte DNA in patients undergoing treatment with cyclophosphamide (11Alarcon R.A. Cancer Treat. Rep. 1976; 60: 327-335PubMed Google Scholar, 12McDiarmid M.A. Iype P.T. Kolodner K. Jacobson-Kram D. Strickland P.T. Mutat. Res. 1991; 248: 93-99Crossref PubMed Scopus (38) Google Scholar). The mutagenic properties of acrolein have been explored in prokaryotic and eukaryotic cells (13Marnett L.J. Hurd H.K. Hollstein M.C. Levin D.E. Esterbauer H. Ames B.N. Mutat. Res. 1985; 148: 25-34Crossref PubMed Scopus (562) Google Scholar, 14Curren R.D. Yang L.L. Conklin P.M. Grafstrom R.C. Harris C.C. Mutat. Res. 1988; 209: 17-22Crossref PubMed Scopus (90) Google Scholar, 15Smith R.A. Cohen S.M. Lawson T.A. Carcinogenesis. 1990; 11: 497-498Crossref PubMed Scopus (51) Google Scholar, 16Kawanishi M. Matsuda T. Nakayama A. Takebe H. Matsui S. Yagi T. Mutat. Res. 1998; 417: 63-75Google Scholar). However, until recently, site-specific mutagenesis studies were not feasible due to the chemical lability of γ-OH-PdG under conditions required for solid phase DNA synthesis. Accordingly, a structural analog, 1,N 2-propano-2′-deoxyguanosine (PdG), which shares the exocyclic ring but lacks the hydroxyl group of naturally occurring acrolein-derived adducts (see Fig. 1), was adopted as a model for structural and biological studies of exocyclic DNA adducts (17Shibutani S. Grollman A.P. J. Biol. Chem. 1993; 268: 11703-11710Abstract Full Text PDF PubMed Google Scholar, 18Hashim M.F. Marnett L.J. J. Biol. Chem. 1996; 271: 9160-9165Abstract Full Text PDF PubMed Scopus (29) Google Scholar, 19Hashim M.F. Schnetz-Boutaud N. Marnett L.J. J. Biol. Chem. 1997; 272: 20205-20212Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 20Benamira M. Singh U. Marnett L.J. J. Biol. Chem. 1992; 267: 22392-22400Abstract Full Text PDF PubMed Google Scholar, 21Moriya M. Zhang W. Johnson F. Grollman A.P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11899-11903Crossref PubMed Scopus (233) Google Scholar, 22Burcham P.C. Marnett L.J. J. Biol. Chem. 1994; 269: 28844-28850Abstract Full Text PDF PubMed Google Scholar, 23Kouchakdjian M. Marinelli E. Gao X.L. Johnson F. Grollman A. Patel D. Biochemistry. 1989; 28: 5647-5657Crossref PubMed Scopus (58) Google Scholar, 24Huang P. Eisenberg M. Biochemistry. 1992; 31: 6518-6532Crossref PubMed Scopus (25) Google Scholar, 25Singh U.S. Moe J.G. Reddy G.R. Weisenseel J.P. Marnett L.J. Stone M.P. Chem. Res. Toxicol. 1993; 6: 825-836Crossref PubMed Scopus (60) Google Scholar, 26Kouchakdjian M. Eisenberg M. Live D. Marinelli E. Grollman A.P. Patel D.J. Biochemistry. 1990; 29: 4456-4465Crossref PubMed Scopus (39) Google Scholar, 27Huang P. Patel D.J. Eisenberg M. Biochemistry. 1993; 32: 3852-3866Crossref PubMed Scopus (16) Google Scholar). Primer extension studies on templates containing a single PdG residue revealed that this adduct induces targeted base substitutions and frameshift mutations in vitro (17Shibutani S. Grollman A.P. J. Biol. Chem. 1993; 268: 11703-11710Abstract Full Text PDF PubMed Google Scholar, 18Hashim M.F. Marnett L.J. J. Biol. Chem. 1996; 271: 9160-9165Abstract Full Text PDF PubMed Scopus (29) Google Scholar, 19Hashim M.F. Schnetz-Boutaud N. Marnett L.J. J. Biol. Chem. 1997; 272: 20205-20212Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). In bacteria, PdG induced frameshift mutations when the lesion was embedded in a CG repeat (20Benamira M. Singh U. Marnett L.J. J. Biol. Chem. 1992; 267: 22392-22400Abstract Full Text PDF PubMed Google Scholar). In other sequence contexts, frameshift mutations were not observed and the principal mutagenic events in bacteria and mammalian cells were G → T transversions and G → A transitions (21Moriya M. Zhang W. Johnson F. Grollman A.P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11899-11903Crossref PubMed Scopus (233) Google Scholar, 22Burcham P.C. Marnett L.J. J. Biol. Chem. 1994; 269: 28844-28850Abstract Full Text PDF PubMed Google Scholar). NMR studies of PdG embedded in duplex DNA showed that, under acidic conditions, the modified base tends to adopt a syn conformation around the glycosyl bond, forming PdG(syn) ·dA+(anti) and PdG(syn)·dC+(anti) alignments. Also, a pH-independent PdG(syn)·dG(anti) base pair was observed in solution (23Kouchakdjian M. Marinelli E. Gao X.L. Johnson F. Grollman A. Patel D. Biochemistry. 1989; 28: 5647-5657Crossref PubMed Scopus (58) Google Scholar, 24Huang P. Eisenberg M. Biochemistry. 1992; 31: 6518-6532Crossref PubMed Scopus (25) Google Scholar, 25Singh U.S. Moe J.G. Reddy G.R. Weisenseel J.P. Marnett L.J. Stone M.P. Chem. Res. Toxicol. 1993; 6: 825-836Crossref PubMed Scopus (60) Google Scholar). In each of these alignments, PdG was inside the helix and hydrogen bonds formed across the base pair involved the Hoogsteen edge of the adduct. An alternative PdG(anti)·dA(anti) alignment, observed at basic pH, showed an adduct exposed to solvent, displaced into the major groove of the helix and unstacked from the flanking bases (26Kouchakdjian M. Eisenberg M. Live D. Marinelli E. Grollman A.P. Patel D.J. Biochemistry. 1990; 29: 4456-4465Crossref PubMed Scopus (39) Google Scholar). The transition between the PdG(syn)·dA+(anti) and PdG(anti)·dA(anti) forms was reversible with a pKa of ∼7.0, indicating that both forms are present and in equilibrium under physiological conditions (26Kouchakdjian M. Eisenberg M. Live D. Marinelli E. Grollman A.P. Patel D.J. Biochemistry. 1990; 29: 4456-4465Crossref PubMed Scopus (39) Google Scholar, 27Huang P. Patel D.J. Eisenberg M. Biochemistry. 1993; 32: 3852-3866Crossref PubMed Scopus (16) Google Scholar). PdG also was used to evaluate the thermodynamic impact of acrolein-derived lesions in DNA duplexes. The adduct reduced thermal stability, transition enthalpy, and transition free energy of the duplex; thermal destabilization was insensitive to the base opposite the adduct (28Plum G.E. Grollman A.P. Johnson F. Breslauer K.J. Biochemistry. 1992; 31: 12096-12102Crossref PubMed Scopus (63) Google Scholar). Recent advances in the chemical synthesis of acrolein-derived adducts (29Khullar S. Varaprasad C.V. Johnson F. J. Med. Chem. 1999; 42: 947-950Crossref PubMed Scopus (49) Google Scholar, 30Nechev L.V. Harris C.M. Harris T.M. Chem. Res. Toxicol. 2000; 13: 421-429Crossref PubMed Scopus (70) Google Scholar) have made it possible to incorporate γ-OH-PdG into oligodeoxynucleotides, enabling the mutagenic properties of this adduct in bacteria to be assessed (see accompanying articles (39Yang I.-Y. Hossain M. Miller H. Khullar S. Johnson F. Grollman A.P. Moriya M. J. Biol. Chem. 2001; 276: 9071-9076Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 40VanderVeen L.A. Hashim M.F. Nechev L.V. Harris T.M. Harris C.M. Marnett L.J. J. Biol. Chem. 2001; 276: 9066-9070Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar)). Surprisingly, synthesis past the adduct was essentially error-free. To better understand the striking differences between the model adduct, PdG, and the naturally occurring acrolein-derived adduct, γ-OH-PdG, we used NMR spectroscopy to determine the solution structure of γ-OH-PdG both as a free nucleoside and in duplex DNA. For the latter structure, the adduct was incorporated opposite dC at the center of an 11-mer oligodeoxynucleotide duplex (referred to as the acr-dG·dC duplex). Our data establish that γ-OH-PdG nucleoside exists in a closed form in solution but undergoes complete conversion to an open structure in duplex DNA. The chemical structure of γ-OH-PdG and the duplex sequence employed in this study are shown in Fig.1. The oligodeoxynucleotide strand containing γ-OH-PdG was synthesized following methods recently described (29Khullar S. Varaprasad C.V. Johnson F. J. Med. Chem. 1999; 42: 947-950Crossref PubMed Scopus (49) Google Scholar). Briefly, theN 2-dihydroxybutyl derivative of dG was introduced into oligomeric DNA by standard phosphoramidite chemical procedures. Sequences containing a terminalO-5′-dimethoxytrityl group were isolated by treatment of the crude synthetic product with concentrated ammonia for 46 h at room temperature and purified by reverse phase HPLC. The mobile phase consisted of solvent A (0.1 m triethylamine acetic acid buffer, pH 6.8) and solvent B (acetonitrile). Using a linear gradient of 0% to 50% of B over 50 min, the desired sequence was eluted as a main fraction at ∼34 min. The O-5′-dimethoxytrityl group was removed by treatment with 80% acetic acid for 30 min, and the solution was extracted with ether three times. TheO-5′-dimethoxytrityl off-products were then purified by HPLC. Subsequent treatment of the oligomer containing theN 2-dihydroxybutyl-dG residue with an excess of an aqueous solution of sodium periodate (0.1 m) at room temperature, until all the starting material disappeared, yielded the desired product. After an additional round of HPLC purification, oligodeoxynucleotide sequences were desalted by passing them through a Sephadex G-25 column and subsequently converted to the sodium salt using a Dowex 50W cation exchange column. Unmodified oligodeoxynucleotide sequences were prepared and purified by standard methods. Electrospray mass spectrometry was used to confirm correct mass/charge ratio of both oligomers. A 1:1 stoichiometry of the duplex was obtained by monitoring the intensity of individual NMR proton signals during gradual addition of the unmodified strand to the γ-(OH)-PdG-containing strand. NMR samples consisted of 130A260 of the duplex dissolved in 0.6 ml of 10 mm phosphate buffer (pH 6.5) containing 50 mmNaCl and 1 mm EDTA in either 99.96% D2O or 90% H2O-10% D2O (v/v), corresponding to a concentration of ∼1.8 mm. Samples of the monomeric γ-OH-PdG nucleoside were dissolved in a similar buffered solution at a final concentration of 0.2 mm. Samples were degassed before collection of the NMR data. One- and two-dimensional NMR spectra were recorded on Varian Inova spectrometers operating at 11.75- and 14.1-Tesla field strengths. Proton chemical shifts were referenced relative to TSP at 0.0 ppm. Phase-sensitive (31States D.J. Habekorn R.A. Ruben D.J. J. Magn. Res. 1982; 48: 286-292Google Scholar) NOESY (120, 200, and 300 ms mixing times), COSY, double quantum filtered-COSY, COSY45, and TOCSY (70- and 120-ms isotropic mixing time) spectra in D2O buffer were collected with a repetition delay of 1.5 s, during which the residual water signal was suppressed by saturation. NOESY spectra (120- and 220-ms mixing time) in 10% D2O buffer were recorded using a jump-return reading pulse (32Plateau P. Gueron M. J. Am. Chem. Soc. 1982; 104: 7310-7311Crossref Scopus (1149) Google Scholar). Time domain data sets consisted of 2048 by 300 complex data points in the t2 and t1dimensions, respectively. For the COSY45 spectrum, 4096 complex points were used in the t2 dimension. NMR data were processed and analyzed using the Felix program (Biosym Technologies, Inc.) running on Silicon Graphics computers. Time domain data sets were multiplied by shifted sinebell window functions prior to Fourier transformation. In the spectra of the free nucleoside, the residual water signal present in the time domain date was eliminated further by subtraction of a fitted polynomial function. No base line correction was applied to the transformed spectra. A three-dimensional model of the acr-dG·dC duplex was built using INSIGHTII (Biosym Technologies, Inc.) by replacing the nonhydrogen-bonded amino proton of a deoxyguanosine residue at the sixth position of a B-form 11-mer duplex for the γ,γ-dihydroxypropyl moiety. Using the conjugate gradient method, this model was energy-minimized to ensure that distances between Hα/Hα′ and Hβ/Hβ′ protons of γ-OH-PdG and the H1′ protons of C7 and G18 residues were within the observable NOE range (see text). Energy minimization was performed on Silicon Graphics computers using the program X-PLOR 3.851 (33Brünger A. X-PLOR, Version 3.1: A system for X-Ray Crystallography and NMR. Yale University Press, New Have, CT1993Google Scholar). At pH values over 6.5, the one-dimensional proton spectrum of the acr-dG·dC duplex displays a main set of sharp signals manageable for NMR characterization. Below this pH value, a second conformation of the duplex in solution is evident by the presence of minor resonances that become stronger as the pH is reduced (see Fig. 5below). Therefore, assignment of the proton signals follows the examination of NOESY and COSY spectra collected at pH 6.5 using standard analysis procedures (34van de Ven J.M. Hilbers C.W. Eur. J. Biochem. 1988; 178: 1-18Crossref PubMed Scopus (290) Google Scholar, 35de los Santos C. Barton D. Nakanishi K. Meth-Cohn O. Comprehensive Natural Products Chemistry, vol 7: DNA and Aspects of Molecular Biology. Elsevier Science Ltd., Oxford, UK1999: 55-80Google Scholar). Fig.2 shows an expanded region of a NOESY spectrum (300-ms mixing time) recorded in 100% D2O buffer at 30 °C, depicting interactions between the base and the H1′ proton regions. Indicative of a right-handed helix, each base proton (purine H8 or pyrimidine H6) shows NOE cross-peaks to the H1′ proton of the ipso and 5′-flanking sugar residues. At the center of the duplex these NOE interactions can be traced without interruption, suggesting that the presence of γ-OH-PdG does not cause large perturbations of the double-helix structure. In addition, the intensity of intra-residue base-H1′ NOE peaks is much weaker than that of the H5-H6 cross-peaks of cytosine residues suggesting an anti-conformation around the glycosydic torsion angle for all residues of the acr-dG·dC duplex (35de los Santos C. Barton D. Nakanishi K. Meth-Cohn O. Comprehensive Natural Products Chemistry, vol 7: DNA and Aspects of Molecular Biology. Elsevier Science Ltd., Oxford, UK1999: 55-80Google Scholar). Additional evidence of a regular right-handed helix is the observation of NOE peaks between each cytosine (H5) and the base proton of its 5′-side neighbor (Fig. 2, peaks A–F). Analogous directionality of NOE interactions is present between the base and sugar H3′, H2′, H2" protons in other regions of the same spectrum (regions not shown). Similarly, nonexchangeable protons of the central (A4 C5 acr-dG C7 A8)·(T15 G16 C17 G18 T19) segment have chemical shift values almost identical to those of the corresponding unmodified control duplex, indicating only a minor deviation from the canonical DNA conformation. Chemical shifts of the nonexchangeable protons of the acr-dG·dC duplex measured at 30 °C are listed in Table I.Figure 2Duplicate contour plots of a portion of the NOESY (300-ms mixing time) spectrum recorded in 100% D2O buffer, pH 6.5, 30 °C. The figure shows distance connectivities between base and H1′ sugar protons in the (left) modified and (right) unmodified strands of the acr-dG·dC duplex.Solid lines connect each base proton (purine H8/pyrimidine H6) to its own (peaks labeled on the figure) and 5′-flanking H1′ sugar protons. Labeled peaks are assigned as follows: A, A4(H8)-C5(H5); B, acr-dG(H8)-C7(H5); C, G10(H8)-C11(H5); D, G12(H8)-C13(H5); E, G16(H8)-C7(H5); F, A20(H8)-C21(H5).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IProton chemical shifts of the acr-dG·dC duplexH6/H8H5/H2/CH3H1′H2′H2"H3′H4′G(NIH)/T(N3H)C(N4H2)C17.665.945.832.032.454.724.088.11 /7.09G27.996.002.702.814.384.0312.81T37.261.535.702.112.454.904.1713.49A48.287.436.232.702.895.044.45C57.245.295.551.962.334.814.177.73 /6.19Acr-dG1-aValues are given in Table II.C77.235.295.611.942.364.864.088.27 /6.43A88.297.596.232.692.925.03NA1-bNA, not assigned.T97.101.465.751.952.374.874.2913.60G107.875.952.612.714.984.3712.76C117.495.506.222.202.204.514.088.17 /6.70G127.966.012.812.634.864.2513.04C137.505.495.732.202.524.924.258.43 /6.61A148.357.766.312.742.985.094.46T157.081.485.751.962.374.884.3113.46G167.825.842.612.695.25NA12.58C177.265.255.781.992.404.84NA8.22 /6.26G187.855.952.762.594.974.3312.66T197.211.505.642.072.404.874.1613.51A208.297.496.212.712.875.044.42C217.285.395.691.872.304.794.168.33 /6.81G227.906.152.382.594.664.1713.04Values are given in parts per million (ppm) relative to TSP. Chemical shifts are recorded in phosphate buffer (10 mm), pH 6.5, containing 50 mm NaCl. Nonexchangeable protons are at 30 °C; exchangeable protons are at 5 °C.1-a Values are given in Table II.1-b NA, not assigned. Open table in a new tab Values are given in parts per million (ppm) relative to TSP. Chemical shifts are recorded in phosphate buffer (10 mm), pH 6.5, containing 50 mm NaCl. Nonexchangeable protons are at 30 °C; exchangeable protons are at 5 °C. Identification of the proton signals of the propyl bridge follows from the analysis of COSY, TOCSY, and NOESY spectra collected in 100% D2O buffer solutions. In the 300-ms mixing time NOESY spectrum, a proton signal at 4.93 ppm, assigned to Hγ, displays NOE cross-peaks to the overlapping Hβ/Hβ′ protons as well as the Hα/Hα′ protons within the propyl moiety (Fig.3 A, peaks A andB, respectively). Accordingly, in a TOCSY (120-ms mixing time) spectrum recorded under identical temperature and pH conditions, cross-peaks are present between these same proton signals (Fig.3 B, peaks A and B, respectively), and among the Hα, Hα′, and Hβ/Hβ′ protons of γ-OH-PdG (region not shown). An intriguing observation is the simultaneous presence of NOE peaks between the Hβ/Hβ′ of the adduct and the H1′ protons of G18 and C7 residues located in opposite strands of the duplex (Fig.3 A, peaks E and C, respectively). Besides this, the presence of a sharp nonexchangeable proton signal is evident at 9.58 ppm, at 30 °C, and slightly upfield at 5 °C (see Fig. 5 below), in a region of the spectrum that is normally devoid of proton signals associated with the duplex. This minor signal shows no cross-peak to any exchangeable or nonexchangeable proton of the duplex and, based on its chemical shift, is assigned to a small percentage of the aldehydic open form of γ-OH-PdG (see Fig. 6 below).Figure 6Chemical rearrangement exerted by γ-(OH)-PdG. The exocyclic form present on the free nucleoside can add a water molecule to afford the hydrated open conformation observed in the acr-dG·dC duplex. Alternatively, chemical rearrangement of γ-OH-PdG produces theN 2-(γ-oxopropyl) configuration of the adduct. The two open forms of the adduct reach equilibrium with the hydrated structure favored at neutral basic solutions.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In the sample dissolved in 10% D2O buffer, the 1D proton spectrum shows 11 imino proton signals resonating between 12.0 and 14.0 ppm, in the Watson-Crick region (see Fig. 5 below). Sequence-specific assignment of the exchangeable proton signals results from the analysis of a NOESY (220-ms mixing time) spectrum collected at 2 °C (pH 6.5). Fig. 4 shows expanded contour plots depicting NOE interactions between the imino and the amino/base proton regions of this spectrum. Each thymine imino proton displays a strong NOE interaction to the H2 proton of the corresponding adenine partner, thus establishing the formation of Watson-Crick alignments for all A·T base pairs of the duplex (Fig. 4, peaks A–D). Similarly, the presence of NOE cross-peaks between the guanine imino and the amino protons of the cytosine partner indicates the formation of Watson-Crick alignments in all nonlesion-containing G·C base pairs of the duplex (Fig. 4, peaks E, E′, I, and I′). Surprisingly, a remaining imino proton signal at 12.64 ppm, which is originated at the acr-dG·dC pair of the duplex, displays strong NOE cross-peaks with three different amino proton signals. Based on interactions to the previously assigned C17(H5) proton and their strong NOE connectivity, which is only observed in 10% D2O buffer, two of these signals are readily assigned to the amino protons of the lesion-partner C17 residue. Thus, peaks J and J′ in Fig. 4 originate from NOE interactions between acr-dG(N1H) imino and C17(N4H2) protons. The third NOE cross-peak originates from the interaction between acr-dG(N1H) and acr-dG(N2H) protons of the adduct (Fig. 4, peak K). These connectivities are only possible when the adduct exists in a ring-opened state so that the lesion-containing base pair adopts the standard Watson-Crick alignment. Consistent with these assignments and supporting the open form of γ-OH-PdG, N1H and N2H display NOE cross-peaks to the Hα/Hα′ and Hβ/Hβ′ protons of the propyl chain (Fig. 4, peaks Q–T). Evidence of base stacking is seen in the connectivities between the adenine H2 protons and the imino protons of the flanking base pairs (Fig. 4, peaks M–O) and those among the imino protons of the duplex (region not shown). Likewise, the strong NOE peak between the amino proton of the adduct and G16(N1H) at the 3′-flanking base pairs indicates proper stacking of γ-OH-PdG inside the duplex (Fig.4, peak L). Chemical shifts of the exchangeable protons of the acr-dG·dC duplex measured at 2 °C are listed in Table I. The unexpected observation that γ-OH-PdG exists in an open form in the duplex prompted us to investigate its state at the nucleoside level. In contrast to observations made with the duplex sample, no proton signals are observed around 9.60 and 4.90 ppm (Fig.5 B). The analysis of a TOCSY spectrum of the nucleoside dissolved in 100% D2O buffer, pH 6.5, 30 °C, reveals that the Hα/Hα′, Hβ/Hβ′, and Hγ protons resonate at 3.52/3.48, 2.22/1.92, and 6.36 ppm, respectively (data not shown). These chemical shift values are slightly downfield from those previously reported for the adduct dissolved in dimethyl sulfoxide (30Nechev L.V. Harris C.M. Harris T.M. Chem. Res. Toxicol. 2000; 13: 421-429Crossref PubMed Scopus (70) Google Scholar, 36Boerth D.W. Eder E. Hussain S. Hoffman C. Chem. Res. Toxicol. 1998; 11: 284-294Crossref PubMed Scopus (16) Google Scholar) and suggest a prevalent closed state for the γ-OH-PdG nucleoside dissolved in water. In addition, the exocyclic form of the adduct is insensitive to pH changes and only the ring-closed state is observed under a wide range of values (Fig.5 B). Upon duplex formation, the chemical shifts of protons on the propyl chain move significantly upfield, especially Hγ, that changes from 6.36 ppm in the nucleoside to 4.93 ppm in the duplex. This chemical shift value, which is inconsistent with the aldehydic proton of the lesion that resonates at 9.58 ppm, is ascribed to the Hγ proton of the propyl chain in which the carbonyl group is present in the hydrated form (dihydroxy) of the adduct (Fig. 6). The relative population of these two forms is dependent on the pH of the sample, the aldehydic form being favored by basic conditions. Apart from these states of γ-(OH)-PdG, an alternative conformation of the acr-dG·dC duplex, which may involve protonated cytosine residues, becomes evident at pH 6.4 and lower values (Fig. 5 A). Proton chemical shifts of the γ-(OH)-1,N 2-PdG nucleoside are listed in Table II.Table IIProton chemical shifts of γ-(HO)-PdGFree nucleosideDuplex DNAH87.887.80Hα/Hα′3.52/3.483.07/3.00Hβ/Hβ′2.22/1.921.72Hγ6.364.93 (9.58)2-aValue on the aldehydic form.N1H12.64N2H7.388.07H1′6.226.04H2′2.452.58H2"2.732.76H3′4.574.97H4′4.064.34H5′/H5"3.77/3.75NA2-bNA, not assigned.Values are given in ppm relative to TSP. Chemical shifts were recorded in phosphate buffer (10 mm), pH 6.5, containing 50 mm NaCl. Nonexchangeable protons are at 30 °C; exchangeable protons are at 5 °C.2-a Value on the aldehydic form.2-b NA, not assigned. Open table in a new tab Values are given in ppm relative to TSP. Chemical shifts were recorded in phosphate buffer (10 mm), pH 6.5, containing 50 mm NaCl. Nonexchangeable protons are at 30 °C; exchangeable protons are at 5 °C. Early in the course of these studies it became evident that the acr-dG·dC duplex adopts a single conformation only at neutral or basic pH (Fig. 5). However, adduct-containing sequences are unstable to the basic conditions used during sample purification, which promote oligomer polymerization (data not shown). Therefore, we chose to conduct our studies at pH 6.5 where ∼85% of the acr-dG·dC duplex is in the conformation present at basic pH. The directionality of sequential NOE interactions indicates that this conformation is a double-stranded helix with residues adopting an anti orientation around the glycosidic bond (Fig. 2). The pattern of NOE peaks observed for the exchangeable imino protons establish that all base pairs of the acr-dG·dC duplex have a Watson-Crick alignment (Fig. 4). At the lesion-containing base pair, this becomes possible only if γ-OH-PdG adduct exists as an open form with the N 2-propyl chain pointing away from the helix and toward the solvent. In this conformation, the Hα/Hα′ and Hβ/Hβ′ protons of γ-OH-PdG are found in the minor groove of the helix, close to H1′ protons of residues in both strands of the duplex (Fig. 3 A,peaks B, C, and D), and its Watson-Crick edge remains accessible forming a fully hydrogen-bonded acr-dG·dC base pair (Fig. 4, peaks J, J′, andK). These structural characteristics are readily fulfilled within a regular B-form helix, as shown by the energy-minimized model of the acr-dG·dC duplex (Fig. 7). Spectroscopic data of the γ-OH-PdG nucleoside in aqueous solutions establish a pH-independent 1,N 2-closed conformation for the adduct (Fig.5 B), suggesting that duplex formation catalyzes the rearrangement of the propyl bridge to an open form. An analogous transformation was described recently for DNA duplexes in which the deoxyguanosine-malondialdehyde adduct M1G is positioned opposite dC (37Mao H. Schnetz-Boutaud N.C. Weisenseel J.P. Marnett L.J. Stone M.P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6615-6620Crossref PubMed Scopus (127) Google Scholar). However, a "canonical" Watson-Crick base pair forms only in the case of γ-OH-PdG. This difference may explain in part why M1G is mutagenic in bacteria (38Fink A.P. Reddy G.R. Marnett L.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8652-8657Crossref PubMed Scopus (157) Google Scholar), whereas γ-OH-PdG is not (see accompanying articles (39Yang I.-Y. Hossain M. Miller H. Khullar S. Johnson F. Grollman A.P. Moriya M. J. Biol. Chem. 2001; 276: 9071-9076Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 40VanderVeen L.A. Hashim M.F. Nechev L.V. Harris T.M. Harris C.M. Marnett L.J. J. Biol. Chem. 2001; 276: 9066-9070Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar)). The role of the partner base in promoting ring opening of γ-OH-PdG adducts will be the subject of future investigations. An unsubstituted 1,N 2-propano-2′-deoxyguanosine adduct has been used extensively in biological (17Shibutani S. Grollman A.P. J. Biol. Chem. 1993; 268: 11703-11710Abstract Full Text PDF PubMed Google Scholar, 18Hashim M.F. Marnett L.J. J. Biol. Chem. 1996; 271: 9160-9165Abstract Full Text PDF PubMed Scopus (29) Google Scholar, 19Hashim M.F. Schnetz-Boutaud N. Marnett L.J. J. Biol. Chem. 1997; 272: 20205-20212Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 20Benamira M. Singh U. Marnett L.J. J. Biol. Chem. 1992; 267: 22392-22400Abstract Full Text PDF PubMed Google Scholar, 21Moriya M. Zhang W. Johnson F. Grollman A.P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11899-11903Crossref PubMed Scopus (233) Google Scholar, 22Burcham P.C. Marnett L.J. J. Biol. Chem. 1994; 269: 28844-28850Abstract Full Text PDF PubMed Google Scholar) and structural (23Kouchakdjian M. Marinelli E. Gao X.L. Johnson F. Grollman A. Patel D. 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Eisenberg M. Biochemistry. 1992; 31: 6518-6532Crossref PubMed Scopus (25) Google Scholar, 25Singh U.S. Moe J.G. Reddy G.R. Weisenseel J.P. Marnett L.J. Stone M.P. Chem. Res. Toxicol. 1993; 6: 825-836Crossref PubMed Scopus (60) Google Scholar). Thesyn conformation permits formation of hydrogen-bonded base pairs through the Hoogsteen edge of the adduct while stacking with flanking residues. Results of the present study establish a fundamental difference between γ-OH-PdG and PdG in that, under appropriate conditions, the former can undergo a chemical rearrangement in aqueous solution to assume an open chain form. Thus, when γ-OH-PdG is in ananti conformation, a fully hydrogen-bonded acr-dG·dC base pair exists at neutral/basic pH values, which does not perturb the duplex structure (Fig. 7). However, at acidic pH, the spectra of the acr-dG·dC duplex show exchangeable proton signals that appear to originate from the amino group of a C+ residue (Fig.5 A). Considering the strong tendency of PdG to adopt asyn conformation, it is likely that, at acidic pH, the duplex contains a syn γ-OH-PdG adduct paired to a protonated cytosine residue forming an alignment similar to the one described for PdG·dC (25Singh U.S. Moe J.G. Reddy G.R. Weisenseel J.P. Marnett L.J. Stone M.P. Chem. Res. Toxicol. 1993; 6: 825-836Crossref PubMed Scopus (60) Google Scholar). The structural characteristics of this conformation in the acr-dG·dC duplex is currently under investigation. Two laboratories have performed primer extension and site-specific mutagenesis studies in bacteria using DNA containing γ-OH-PdG. Synthesis past the lesion is reduced indicating that γ-OH-PdG blocks DNA synthesis and, when translesional synthesis occurs, dCMP is incorporated opposite the lesion almost exclusively (see accompanying articles (39Yang I.-Y. Hossain M. Miller H. Khullar S. Johnson F. Grollman A.P. Moriya M. J. Biol. Chem. 2001; 276: 9071-9076Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 40VanderVeen L.A. Hashim M.F. Nechev L.V. Harris T.M. Harris C.M. Marnett L.J. J. Biol. Chem. 2001; 276: 9066-9070Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar)). The present study provides structural grounds for understanding this behavior. At the replication fork γ-OH-PdG would adopt the closed 1,N 2-exocyclic form described for the free nucleoside in solution. As with PdG, this conformation of the adduct is expected to hinder incorporation of dAMP, dGMP, and TMP, resulting in the inhibition of DNA synthesis. However, incorporation of dCMP opposite γ-OH-PdG would trigger the chemical rearrangement from the exocyclic closed form of the adduct to an opened conformation. The subsequent formation of a replication structure stabilized by Watson-Crick hydrogen bonds would facilitate rapid extension of the γ-OH-PdG·dC pair resulting in error-free translesional DNA synthesis. Thus, chemical rearrangement of γ-OH-PdG to an open form during DNA synthesis would account for the lack of mutagenicity observed with the major acrolein-derived 2′-deoxyguanosine adduct in bacteria. We thank Cecilia Torres for the synthesis and purification of modified oligodeoxynucleotides and Arthur P. Grollman for critical reading of this manuscript.

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