Structure of poly d(AI).poly d(CT) in two different packing arrangements

X-ray diffraction analysis of poly d(AI).poly d(CT) in oriented and polycrystalline fibers has revealed the DNA structure to be a 10-fold, right-handed, antiparallel, Watson-Crick base paired double helix in two distinct packing arrangements corresponding to one and two helices, respectively, in the unit cell. The helix pitch is 32.1 A and 32.4 A in the two cases, almost 1.5 A shorter than in classical B-DNA. The resulting B'-DNA geometry, described in terms of a tetranucleotide repeat which is conformationally similar to B-DNA, has its minor groove closely shut and major groove correspondingly widened, thus striking a sharp morphological contrast to B-DNA. According to difference electron density maps, a spine of hydration along the minor groove connects both strands and provides structural stability; ordered sodium ions and water molecules are actively involved in bridging the phosphate groups of neighboring helices. The crystallographic R-values for these two allomorphs are 0.26 and 0.20, respectively, for data up to 3.0 A resolution.     

Minor groove binding of a bis-quaternary ammonium compound: the crystal structure of SN 7167 bound to d(CGCGAATTCGCG)2

The X-ray crystal structure of the complex between the synthetic antitumour and antiviral DNA binding ligand SN 7167 and the DNA oligonucleotide d(CGCGAATTCGCG)2 has been determined to an R factor of 18.3% at 2.6 A resolution. The ligand is located within the minor groove and covers almost 6 bp with the 1-methylpyridinium ring extending as far as the C9-G16 base pair and the 1-methylquinolinium ring lying between the G4-C21 and A5-T20 base pairs. The ligand interacts only weakly with the DNA, as evidenced by long range contacts and shallow penetration into the groove. This structure is compared with that of the complex between the parent compound SN 6999 and the alkylated DNA sequence d(CGC[e6G]AATTCGCG)2. There are significant differences between the two structures in the extent of DNA bending, ligand conformation and groove binding.     

Divalent transition metal cations counteract potassium-induced quadruplex assembly of oligo(dG) sequences

Nucleic acids containing tracts of contiguous guanines tend to self-associate into four-stranded (quadruplex) structures, based on reciprocal non-Watson-Crick (G*G*G*G) hydrogen bonds. The quadruplex structure is induced/stabilized by monovalent cations, particularly potassium. Using circular dichroism, we have determined that the induction/stabilization of quadruplex structure by K+is specifically counteracted by low concentrations of Mn2+(4-10 mM), Co2+(0.3-2 mM) or Ni2+(0.3-0.8 mM). G-Tract-containing single strands are also capable of sequence-specific non-Watson-Crick interaction with d(G. C)-tract-containing (target) sequences within double-stranded DNA. The assembly of these G*G.C-based triple helical structures is supported by magnesium, but is potently inhibited by potassium due to sequestration of the G-tract single strand into quadruplex structure. We have used DNase I protection assays to demonstrate that competition between quadruplex self-association and triplex assembly is altered in the presence of Mn2+, Co2+or Ni2+. By specifically counteracting the induction/stabilization of quadruplex structure by potassium, these divalent transition metal cations allow triplex formation in the presence of K+and shift the position of equilibrium so that a very high proportion of triplex target sites are bound. Thus, variation of the cation environment can differentially promote the assembly of multistranded nucleic acid structural alternatives.     

A molecular mechanics and dynamics study of the minor adduct between DNA and the carcinogen 2-(acetylamino)fluorene (dG-N2-AAF)

Experimental studies involving the carcinogenic aromatic amine 2-(acetylamino)fluorene (AAF) have afforded two acetylated DNA adducts, the major one bound to C8 of guanine and a minor adduct bound to N2 of guanine. The minor adduct may be important in carcinogenesis because it persists, while the major adduct is rapidly repaired. Primer extension studies of the minor adduct have indicated that it blocks DNA synthesis, with some bypass and misincorporation of adenine opposite the lesion [Shibutani, S., and Grollman, A.P. (1993) Chem. Res. Toxicol. 6, 819-824]. No experimental structural information is available for this adduct. Extensive minimized potential energy searches involving thousands of trials and molecular dynamics simulations were used to study the conformation of this adduct in three sequences: I, d(C1-G2-C3-[AAF]G4-C5-G6-C7).d(G8-C9-G10-C11-G12-C13-G14+ ++); II, the sequence of Shibutani and Grollman, d(C1-T2-A3-[AAF]G4-T5-C6-A7).d(T8-G9-A10-C11-T12-A13-G14); and III, which is the same as II but with a mismatched adenine in position 11, opposite the lesion. AAF was located in the minor groove in the low-energy structures of all sequences. In the lowest energy form of the C3-[AAF]G4-C5 sequence I, the fluorenyl rings point in the 3' direction along the modified strand and the acetyl in the 5' direction. These orientations are reversed in the second lowest energy structure of this sequence, and the energy of this structure is 1.4 kcal/mol higher. Watson Crick hydrogen bonding is intact in both structures. In the two lowest energy structures of the A3-[AAF]G4-T5 sequence II, the AAF is also located in the minor groove with Watson-Crick hydrogen bonding intact. However, in the lowest energy form, the fluorenyl rings point in the 5' direction and the acetyl in the 3' direction. The energy of the structure with opposite orientation is 5.1 kcal/mol higher. In sequence III with adenine mismatched to the modified guanine, the lowest energy form also had the fluorenyl rings oriented 5' in the minor groove with intact Watson-Crick base pairing. However, the mispaired adenine adopts a syn orientation with Hoogsteen pairing to the modified guanine. These results suggest that the orientation of the AAF in the minor groove may be DNA sequence dependent. Mobile aspects of favored structures derived from molecular dynamics simulations with explicit solvent and salt support the essentially undistorting nature of this lesion, which is in harmony with its persistence in mammalian systems.     

RNA hydration: a detailed look

The crystal structure of the RNA duplex [r(CCCCGGGG)]2 has been refined to 1.46 A resolution with room temperature synchrotron diffraction data. This represents the highest resolution reported to date for an all-RNA oligonucleotide and is well beyond the best resolution ever achieved with an A-form DNA duplex. The analysis of the ordered hydration around the octamer duplex reveals conserved regular arrangements of water molecules in both grooves. In the major groove, all located first shell water molecules can be fitted into a pattern that is repeated through all eight base pairs, involves half the phosphate oxygens, and joins the two strands. In the minor groove, roughly across its narrowest dimension, tandem water molecules link the 2'-hydroxyl groups of adjacent nucleotides in base-pair steps in a similarly regular fashion. The structure provides evidence for an important role of the 2'-hydroxyl groups in the thermodynamic stabilization of RNA, beyond their known functions of locking the sugar pucker and mediating 3' --> 5' intrastrand O2'...O4' hydrogen bonds. The ribose 2'-hydroxyls lay the foundation for the enthalpic stability of the RNA relative to the DNA duplex, both as a scaffold for the water network in the minor groove and through their extensive individual hydration.     

Catalytic role of monovalent cations in the mechanism of proton transfer which gates an interprotein electron transfer reaction

Within the methylamine dehydrogenase (MADH)-amicyanin protein complex, long range intermolecular electron transfer (ET) occurs between tryptophan tryptophylquinone (TTQ) of MADH and the type I copper of amicyanin. The reoxidations of two chemically distinct reduced forms of TTQ were studied, a quinol (O-quinol) generated by reduction by dithionite and the physiologically relevant aminoquinol (N-quinol) generated by reduction by methylamine. The latter contains a substrate-derived amino group which displaces the C6 carbonyl oxygen on TTQ. ET from N-quinol MADH to amicyanin is gated by the transfer of a solvent exchangeable proton [Bishop, G. R., & Davidson, V. L. (1995) Biochemistry 34, 12082-12086]. The factors which influence this proton transfer (PT) reaction have been examined. The rate of PT increases with increasing pH and with increasing salt concentration. The salt effect is due to specific monovalent cations and is not a general ionic strength effect. The rate enhancements by pH and cations do not reflect an elimination of the PT step that gates ET. Over the range of pH from 5.5 to 9.0 and with cation concentrations from 0 to 200 mM, the observed rate of the redox reaction is still that of PT. This is proven by kinetic solvent isotope effect studies which show that a primary isotope effect persists even at the highest values of pH and cation concentration. A model is presented to explain how specific cations contribute to catalysis and influence the rate of PT in this reaction. The pH dependence is attributed to an ionizable group that is involved in cation binding. The effect of the cation is stabilization of a negatively charged reaction intermediate that is formed during the deprotonation of the N-quinol, and from which rapid ET to the copper of amicyanin occurs. The relevance of these findings to other enzymes which exhibit reaction rates that are influenced by monovalent cations is also discussed.     

Divalent cations stabilize unstacked conformations of DNA and RNA by interacting with base pi systems

Nucleic acid structure, stability, and reactivity are governed substantially by cations. We propose that magnesium and other biological inorganic ions unstack bases of DNA and RNA. This unstacking function of cations opposes their previously accepted role in stabilizing DNA and RNA duplexes and higher assemblies. We show that cations interact favorably with pi-systems of nucleic acid bases. These cation-pi interactions require access of cations or their first hydration shells to faces of nucleic acid bases. We observe that hydrated magnesium ions located in the major groove of B-DNA pull cytosine bases partially out from the helical stack, exposing pi-systems to positive charge. A series of critical cation-pi interactions contribute to the stability of the anticodon arm of yeast-tRNAphe, and to the magnesium core of the Tetrahymena group I intron P4-P6 domain. The structural consequences of divalent cation-pi interactions are clearly distinct from, and some cases in opposition to, cation-electron lone pair interactions. These observations of cation-pi interactions suggest a number of new mechanistic roles for cations in DNA bending, DNA-protein recognition, base-flipping, RNA folding, and catalysis.     

Hin recombinase bound to DNA: the origin of specificity in major and minor groove interactions

The structure of the 52-amino acid DNA-binding domain of the prokaryotic Hin recombinase, complexed with a DNA recombination half-site, has been solved by x-ray crystallography at 2.3 angstrom resolution. The Hin domain consists of a three-alpha-helix bundle, with the carboxyl-terminal helix inserted into the major groove of DNA, and two flanking extended polypeptide chains that contact bases in the minor groove. The overall structure displays features resembling both a prototypical bacterial helix-turn-helix and the eukaryotic homeodomain, and in many respects is an intermediate between these two DNA-binding motifs. In addition, a new structural motif is seen: the six-amino acid carboxyl-terminal peptide of the Hin domain runs along the minor groove at the edge of the recombination site, with the peptide backbone facing the floor of the groove and side chains extending away toward the exterior. The x-ray structure provides an almost complete explanation for DNA mutant binding studies in the Hin system and for DNA specificity observed in the Hin-related family of DNA invertases.     

Determination of the residence time of water molecules hydrating B'- DNA and B-DNA, by one-dimensional zero-enhancement nuclear Overhauser effect spectroscopy

The residence time of water in the minor groove of the d(CGCGAATTCGCG) duplex has been determined by a recent measurement combining nuclear Overhauser enhancements (NOE, ROE) and 17O relaxation dispersion. The time is in the range of nanoseconds, so that it may be measured by a rather simple method proposed here, namely the choice of conditions such that the NOE between the observed DNA proton and a nearby water proton is zero. This condition is realized when the residence time of the water molecule is 0.178 times the nuclear magnetic resonance period (e.g. 0.297 ns at 600 MHz). It may be achieved by varying the magnetic field and/or the temperature. The zero-NOE measurement may be performed by one-dimensional NMR, and has therefore good sensitivity. We have developed excitation sequences which suppress two spurious contributions to the NOE: from neighboring exchangeable protons and from H3' protons whose chemical shift is close to that of water. The method is applied here to the comparison of residence times of water next to B-DNA and next to B'-DNA, the latter corresponding to better stacked, propeller-twisted base-pairs and a correspondingly narrower minor groove. In the minor groove of [d(CGCGAATTCGCG)]2, a B'-DNA duplex, the residence time of the water molecule next to H2 of adenine(6) (underlined), is 0.6 ns at 10 degreesC, in good agreement with the value obtained previously. The residence time is slightly but distinctly shorter for the water next to A5, suggesting non-cooperative departure of these two molecules which are presumed to be part of the hydration spine. Near A5 and A4 of [d(AAAAATTTTT)]2, another B'-DNA duplex, the residence times are approximately twice as long, but the activation enthalpies are about the same, ca. 38 kJ/mol. The residence time in the minor groove of the regular B-DNA sequence d(CGCGATCGCG) was 0.3 ns at 10 degreesC, shorter than in the case of the B'-DNA sequences by factors of 2 and 4, respectively. The temperature dependence is less, with an activation enthalpy of 27 kJ/mol. The major groove residence times are comparable for the three sequences, and a few times shorter than those of minor groove water. A value of 0.36 ns, or even more in case of rotation of water, is obtained around -8 degreesC. The most striking aspect of these results is the relatively small difference in the residence times of reputedly fast and slow-exchanging water molecules bound to DNA in biological conditions. This suggests that the spine of hydration is perhaps not a major stabilizer of the B'-DNA structure as compared with B-DNA.     

The high resolution crystal structure of the DNA decamer d(AGGCATGCCT)

The crystal structure of the DNA decamer d(AGGCATGCCT) has been determined to a resolution of 1.3 A and R factor of 13.9%. The structure has a unique conformation with each of the decamer single strands forming base-pairing interactions with two symmetry-related strands. The central eight bases of the decamer form an A-DNA octamer duplex with one symmetry-related strand whilst the terminal 5'-A and T-3' bases are flipped out and away from the octamer helix axis to form base-pairing interactions with a second symmetry-related strand. These A.T base-pairs lie perpendicular to the crystallographic c axis and pack within the unit cell in conjunction with a symmetry-related A.T base-pair displaced by 3.4 A degrees along the c axis. A novel base triplet interaction of the type A*(G.C) is present in the structure with interaction from the major groove side of the terminal 5'-A base to the minor groove of the central A-DNA octamer. This structure reports the first example of cobalt hexammine binding to a right-handed DNA duplex. The crystallographic asymmetric unit contains two cobalt hexammine ligands with one site in the major groove coordinating via hydrogen bonds to the 5'-AGG bases, and the second site located between DNA molecules and interacting with the oxygen atoms of phosphate groups.     

Structure-based discovery of ligands targeted to the RNA double helix

Ligands capable of specific recognition of RNA structures are of interest in terms of the principles of molecular recognition as well as potential chemotherapeutic applications. We have approached the problem of identifying small molecules with binding specificity for the RNA double helix through application of the DOCK program [Kuntz, I. D., Meng, E. C., and Shoichet, B. K. (1994) Acc. Chem. Res. 27, 117-123], a structure-based method for drug discovery. A series of lead compounds was generated through a database search for ligands with shape complementarity to the RNA deep major groove. Compounds were then evaluated with regard to their fit into the minor groove of B DNA. Those compounds predicted to have an optimal fit to the RNA groove and strong discrimination against DNA were examined experimentally. Of the 11 compounds tested, 3, all aminoglycosides, exhibited pronounced stabilization of RNA duplexes against thermal denaturation with only marginal effects on DNA duplexes. One compound, lividomycin, was examined further, and shown to facilitate the ethanol-induced B to A transition in calf thymus DNA. Fluorine NMR solvent isotope shift measurements on RNA duplexes containing 5-fluorouracil provided evidence that lividomycin binds in the RNA major groove. Taken together, these results indicate that lividomycin recognizes the general features of the A conformation of nucleic acids through deep groove binding, confirming the predictions of our DOCK analysis. This approach may be of general utility for identifying ligands possessing specificity for additional RNA structures as well as other nucleic acid structural motifs.     

Minor groove-directed and intercalative ligand-DNA interactions in the poisoning of human DNA topoisomerase I by protoberberine analogs

Spectroscopic, calorimetric, DNA cleavage, electrophoretic, and computer modeling techniques have been employed to characterize the DNA binding and topoisomerase poisoning properties of three protoberberine analogs, 8-desmethylcoralyne (DMC), 5,6-dihydro-8-desmethylcoralyne (DHDMC), and palmatine, which differ in the chemical structures of their B- and/or D-rings. DNA topoisomerase-mediated cleavage assays revealed that these compounds were unable to poison mammalian type II topoisomerase. By contrast, the three protoberberine analogs poisoned human topoisomerase I according to the following hierarchy: DHDMC > DMC > palmatine. DNA binding by all three protoberberine analogs induced negative flow linear dichroism signals as well as unwinding of the host duplex. These two observations are consistent with an intercalative mode of protoberberine binding to duplex DNA. However, a comparison of the DNA binding properties for DMC and DHDMC, which differ only by the state of saturation at the 5,6 positions of the B-ring, revealed that the protoberberine analogs do not "behave" like classic DNA intercalators. Specifically, saturation of the 5-6 double bond in the B-ring of DMC, thereby converting it to the DHDMC molecule, was associated with enhanced DNA unwinding as well as a reversal of DNA binding preference from a DNA duplex with an inaccessible or occluded minor groove {poly[d(G-C)]2} to DNA duplexes with accessible or unobstructed minor grooves {poly[d(A-T)]2 and poly[d(I-C)]2}. In addition, a comparison of the DNA binding properties for DHDMC and palmatine revealed that transferring the 11-methoxy moiety on the D-ring of DHDMC to the 9 position, thereby converting it to palmatine, was associated with a reduction in binding affinity for both duplexes with unobstructed minor grooves as well as for duplexes with occluded minor grooves. These DNA binding properties are consistent with a "mixed-mode" DNA binding model for protoberberines in which a portion of the ligand molecule intercalates into the double helix, while the nonintercalated portion of the ligand molecule protrudes into the minor groove of the host duplex, where it is thereby available for interactions with atoms lining the floor and/or walls of the minor groove. Furthermore, saturation at the 5,6 positions of the B-ring, which causes the A-ring to be tilted relative to the plane formed by rings C and D, appears to stabilize the interaction between the host duplex and the minor groove-directed portion of the protoberberine ligand. Computer modeling studies on the DHDMC-poly[d(A-T)]2 complex suggest that this interaction may involve van der Waals contacts between the ligand A-ring and backbone sugar atoms lining the minor groove of the host duplex. The hierarchy of topoisomerase I poisoning noted above suggests that this minor groove-directed interaction may play an important role in topoisomerase I poisoning by protoberberine analogs. In the aggregate, our results presented here, coupled with the recent demonstration of topoisomerase I poisoning by minor groove-binding terbenzimidazoles [Sun, Q., Gatto, B., Yu, C., Liu, A. , Liu, L. F., & LaVoie, E. J. (1995) J. Med. Chem. 38, 3638-3644], suggest that minor groove-directed ligand-DNA interactions may be of general importance in the poisoning of topoisomerase I.     

Structural basis of DNA folding and recognition in an AMP-DNA aptamer complex: distinct architectures but common recognition motifs for DNA and RNA aptamers complexed to AMP

BACKGROUND: Structural studies by nuclear magnetic resonance (NMR) of RNA and DNA aptamer complexes identified through in vitro selection and amplification have provided a wealth of information on RNA and DNA tertiary structure and molecular recognition in solution. The RNA and DNA aptamers that target ATP (and AMP) with micromolar affinity exhibit distinct binding site sequences and secondary structures. We report below on the tertiary structure of the AMP-DNA aptamer complex in solution and compare it with the previously reported tertiary structure of the AMP-RNA aptamer complex in solution. RESULTS: The solution structure of the AMP-DNA aptamer complex shows, surprisingly, that two AMP molecules are intercalated at adjacent sites within a rectangular widened minor groove. Complex formation involves adaptive binding where the asymmetric internal bubble of the free DNA aptamer zippers up through formation of a continuous six-base mismatch segment which includes a pair of adjacent three-base platforms. The AMP molecules pair through their Watson-Crick edges with the minor groove edges of guanine residues. These recognition G.A mismatches are flanked by sheared G.A and reversed Hoogsteen G.G mismatch pairs. CONCLUSIONS: The AMP-DNA aptamer and AMP-RNA aptamer complexes have distinct tertiary structures and binding stoichiometries. Nevertheless, both complexes have similar structural features and recognition alignments in their binding pockets. Specifically, AMP targets both DNA and RNA aptamers by intercalating between purine bases and through identical G.A mismatch formation. The recognition G.A mismatch stacks with a reversed Hoogsteen G.G mismatch in one direction and with an adenine base in the other direction in both complexes. It is striking that DNA and RNA aptamers selected independently from libraries of 10(14) molecules in each case utilize identical mismatch alignments for molecular recognition with micromolar affinity within binding-site pockets containing common structural elements.     

Efficient priming of PCR with short oligonucleotides conjugated to a minor groove binder

The tripeptide 1,2-dihydro-(3H)-pyrrolo[3,2-e]indole-7-carboxylate (CDPI3) binds to the minor groove of DNA with high affinity. When this minor groove binder (MGB) is conjugated to the 5'-end of short oligodeoxynucleotides (ODNs), the conjugates form unusually stable hybrids with complementary DNA in which the tethered CDPI3group resides in the minor groove. We show that these conjugates can be used as PCR primers. Due to their unusually high binding affinity, conjugates as short as 8-10mers can be used to amplify DNA with good specificity and efficiency. The reduced length primers described here might be appropriate for the PCR amplification of viral sequences which possess a high degree of variability (e.g., HPV, HIV) or for recent techniques such as gene hunting and differential display which amplify multiple sequences using short primer pairs.     

Crystal structure of LacI member, PurR, bound to DNA: minor groove binding by alpha helices

The three-dimensional structure of a ternary complex of the purine repressor, PurR, bound to both its corepressor, hypoxanthine, and the 16-base pair purF operator site has been solved at 2.7 A resolution by x-ray crystallography. The bipartite structure of PurR consists of an amino-terminal DNA-binding domain and a larger carboxyl-terminal corepressor binding and dimerization domain that is similar to that of the bacterial periplasmic binding proteins. The DNA-binding domain contains a helix-turn-helix motif that makes base-specific contacts in the major groove of the DNA. Base contacts are also made by residues of symmetry-related alpha helices, the "hinge" helices, which bind deeply in the minor groove. Critical to hinge helix-minor groove binding is the intercalation of the side chains of Leu54 and its symmetry-related mate, Leu54', into the central CpG-base pair step. These residues thereby act as "leucine levers" to pry open the minor groove and kink the purF operator by 45 degrees.     

1H NMR study of [d(GCGATCGC)]2 and its interaction with minor groove binding 4',6-diamidino-2-phenylindole

Two-dimensional NMR spectroscopy has been applied to study the solution binding of 4',6-diamidino-2-phenylindole (DAPI) to synthetic DNA duplex [d(GCGATCGC)]2. The structure of the complex at a molar ratio of 1:1 drug:duplex has been investigated. NMR results indicate that DAPI binds selectively in the minor groove of the DNA region containing only two A:T base pairs. The results disagree with conclusions drawn from footprinting experiments and show that the presence of the G3NH2 group in the minor groove does not prevent the binding. A molecular model is proposed that closely resembles the crystal structure previously published for the interaction of DAPI with the dodecamer [d(CGCGAATTCGCG)]2, containing four A:T base pairs in the binding site. In this model, DAPI lies in the minor groove, nearly isohelical, with its aromatic rings adjacent to H4' protons of T5 and C6 deoxyribose and the NH indole group oriented toward the DNA axis. The binding does not perturb the B-type conformation of the duplex, and the DNA oligomer conserves its 2-fold symmetry, indicating that fast exchange dynamics exist between the two stereochemically equivalent binding sites of the palindromic sequence. The binding constant and the exchange rate between free and bound species were also measured by NMR spectroscopy.     

GC rich DNA oligonucleotides with narrow minor groove width

Investigation of the width of the minor groove using 500 MHz NMR spectroscopy in three closely related 11-mer B-DNA duplexes shows that the minor groove is narrow in a GC rich oligonucleotide, and that a narrow minor groove is not something endemic to DNAs with persistent repetitions of adenine nucleotides (A-tract DNA). The width of the groove is dictated by local sequence contexts and independent of neighboring A-tract DNA.     

Improved targeting of an anti-epidermal growth factor receptor variant III monoclonal antibody in tumor xenografts after labeling using N-succinimidyl 5-iodo-3-pyridinecarboxylate

The crystal structure is reported of a complex between the dodecanucleotide sequence d(CGCGAATTCGCG)2and an analogue of the DNA binding drug Hoechst 33258, in which the piperazine ring has been replaced by an amidinium group and the phenol ring by a phenylamidinium group. The structure has been refined to an R factor of 19.5% at 2.2 A resolution. The drug is held in the minor groove by five strong hydrogen bonds, together with bridging water molecules at both ends. There are few other contacts with the floor of the groove, indicating a lack of isohelicity with the groove and suggesting (i) that the observed high DNA affinity of this drug is primarily due to the array of hydrogen bonds and (ii) that these more than compensate for its poor isohelicity.     

Structural effects of the C2-methylhypoxanthine:cytosine base pair in B-DNA: A combined NMR and X-ray diffraction study of d(CGC[m2I]AATTCGCG)

C2-Methylhypoxanthine (m2I) is a synthetic analog of guanine with the N2-amino group replaced by a methyl group. We have studied the structural consequence of the m2I incorporation in DNA by a combination of X-ray crystallographic, NMR, and enzymatic analyses. The crystal structure of d(CGC[m2I]AATTCGCG) has been solved and refined to an R factor of 20.7% at 2.25-A resolution. In the DNA duplex, the two independent m2I:C base pairs maintain the Watson-Crick scheme. While the C2-methyl group of m2I is in van der Waals contact with the O2 of the base-paired cytosine, it only causes the base pair to have slightly higher propeller twist and buckle angles. Its solution structure was analyzed by the NMR refinement procedure SPEDREF [Robinson, H., & Wang, A. H.-J. (1992) Biochemistry 31, 3524-3533] using 2D nuclear Overhauser effect data. Two starting models, a relaxed fiber model and an X-ray model, were subjected to the NOE-constrained refinement using 1518 NOE cross-peak integrals to arrive at the final models with (NOE) R factors of 13.8% and 14.3%, respectively. The RMSD between the two refined models (all atoms included) is 1.23 A, which presently seems to be near the limit of convergence of NOE-based refinement. The local structures of the two models are in better agreement as measured by the RMSD of the dinucleotide steps, falling in the range 0.54-0.98 A. Both refined solution structures confirm that the m2I dodecamer structure is of the B-DNA type with a narrow minor groove at the AT region, as observed in the crystal. However, significant differences exist between the crystal and solution structures in parameters such as pseudorotation angles, propeller twist angles, etc. The solution structure tends to have a more uniform backbone conformation, an observation consistent with that concluded from the laser Raman study of d(CGCAAATTTGCG) [Benevides, J. M., Wang, A. H.-J., van der Marel, G. A., van Boom, J. H., & Thomas, G. J., J. (1988) Biochemistry 27, 931-938]. Three related dodecamers, d(CGCGAATTCGCG), d(CGC[m2I]AATTCGCG), and d(CGC[e6G]AATTCGCG), were tested as substrates for the restriction endonuclease EcoRI. The m2I dodecamer was active, but the e6G dodecamer was not. Our results illustrate the complementarity in terms of the structural information provided by the two methods, X-ray diffraction and NMR.