DNA interstrand crosslink formation by mechlorethamine at a cytosine-cytosine mismatch pair: kinetics and sequence dependence

Expansion of the triplet repeat DNA sequence d[CGG]n.d[CCG]n is a characteristic of Fragile X syndrome, a human neurodegenerative disease. Stable intrastrand conformations formed by both d[CGG]n and d[CCG]n, and involving G-G and C-C mismatch pairs, respectively, are believed to be of importance in the development of the disease. We have shown previously that C-C mismatch pairs can be crosslinked covalently by mechlorethamine, a nitrogen mustard alkylating agent, and hence this reaction may be of value as a probe for conformers of d[CCG]n. To characterize the mechlorethamine C-C crosslink reaction further, here we report the kinetics and sequence dependence of formation of the crosslink species, using a series of model duplexes. The rate of reaction depends on the base sequence proximal to the C-C mismatch pair. Hence, in 19mer duplexes containing a central d[M4M3M2M1Cn1n2n3n4].d[N4N3N2N1Cm1m2m3m4] sequence, where M-m and N-n are complementary base pairs, the amount of crosslink increased with increasing G-C content of the eight base pairs neighboring the C-C mismatch and with the proximity of the G-C pairs to the C-C mismatch. Molecular dynamics simulations of the solvated duplexes provided an explanation of these data. Hence, for a C-C pair flanked by G-C base pairs the mismatched cytosine bases remain stacked within the duplex, but for a C-C pair flanked by A-T base pairs, the simulations suggested local opening of the duplex around the C-C pair, making it a less effective target for mechlorethamine.     

Solution structure of a DNA duplex containing mispair-aligned N4C-ethyl-N4C interstrand cross-linked cytosines

The solution structure of an interstrand cross-linked self-complementary oligodeoxynucleotide containing directly opposed alkylated N(4)C-ethyl-N(4)C cytosine bases was determined by molecular dynamics calculations guided by NMR-derived restraints. The undecamer d(CGAAACTTTCG)(2), where C represents directly opposed alkylated N(4)C-ethyl-N(4)C cytosine bases, serves as model for the cytotoxic cross-links formed by bifunctional alkylating agents used in cancer therapy. The structure of the duplex shows the cross-link protruding into the major groove. An increase in the diameter of the DNA at the pseudoplatform formed by the cross-linked residues creates an A-DNA characteristic hole in the central portion of the DNA. This results in a centrally underwound base step and a number of subsequent overwinding steps leading to an overall axis bend toward the major groove. The structure shows narrowing of both minor and major grooves in the proximity of the cross-link. The perturbation leads to preferential intrastrand base stacking, disruption of adjacent canonical (A.T) base pairing, and buckling of base pairs, the extent of which diminishes with progression away from the lesion site. Overall, the distortion induced by the cross-link spreads over three base pairs on the 5'- and 3'-sides of the cross-link.     

Solution structure of a nitrous acid induced DNA interstrand cross-link

Nitrous acid is a mutagenic agent. It can induce interstrand cross-links in duplex DNA, preferentially at d(CpG) steps: two guanines on opposite strands are linked via a single shared exocyclic imino group. Recent synthetic advances have led to the production of large quantities of such structurally homogenous cross-linked duplex DNA. Here we present the high resolution solution structure of the cross-linked dodecamer [d(GCATCCGGATGC)]2 (the cross-linked guanines are underlined), determined by 2D NMR spectroscopy, distance geometry, restrained molecular dynamics and iterative NOE refinement. The cross-linked guanines form a nearly planar covalently linked 'G:G base pair' with only minor propeller twisting, while the cytidine bases of their normal base pairing partners have been flipped out of the helix and adopt well defined extrahelical positions in the minor groove. On the 5'-side of the cross-link, the minor groove is widened to accommodate these extrahelical bases, and the major groove becomes quite narrow at the cross-link. The cross-linked 'G:G base pair' is well stacked on the spatially adjacent C:G base pairs, particularly on the 3'-side guanines. In addition to providing the first structure of a nitrous acid cross-link in DNA, these studies could be of major importance to the understanding of the mechanisms of nitrous acid cross-linking and mutagenicity, as well as the mechanisms responsible for its repair in intracellular environments. It is also the shortest DNA cross-link structure to be described.     

N(4)C-ethyl-N(4)C cross-linked DNA: synthesis and characterization of duplexes with interstrand cross-links of different orientations.

The preparation and physical properties of short DNA duplexes that contain a N(4)C-ethyl-N(4)C interstrand cross-link are described. Duplexes that contain an interstrand cross-link between mismatched C-C residues and duplexes in which the C residues of a -CG- or -GC- step are linked to give "staggered" interstrand cross-links were prepared using a novel N(4)C-ethyl-N(4)C phosphoramidite reagent. Duplexes with the C-C mismatch cross-link have UV thermal transition temperatures that are 25 degrees C higher than the melting temperatures of control duplexes in which the cross-link is replaced with a G-C base pair. It appears that this cross-link stabilizes adjacent base pairs and does not perturb the structure of the helix, a conclusion that is supported by the CD spectrum of this duplex and by molecular models. An even higher level of stabilization, 49 degrees C, is seen with the duplex that contains a -CG- staggered cross-link. Molecular models suggest that this cross-link may induce propeller twisting in the cross-linked base pairs, and the CD spectrum of this duplex exhibits an unusual negative band at 298 nm, although the remainder of the spectrum is similar to that of B-form DNA. Mismatched C-C or -CG- staggered cross-linked duplexes that have complementary overhanging ends can undergo self-ligation catalyzed by T4 DNA ligase. Analysis of the ligated oligomers by nondenaturing polyacrylamide gel electrophoresis shows that the resulting oligomers migrate in a manner similar to that of a mixture of non-cross-linked control oligomers and suggests that these cross-links do not result in significant bending of the helix. However, the orientation of the staggered cross-link can have a significant effect on the structure and stability of the cross-linked duplex. Thus, the thermal stability of the duplex that contains a -GC- staggered cross-link is 10 degrees C lower than the melting temperature of the control, non-cross-linked duplex. Unlike the -CG- staggered cross-link, in which the cross-linked base pairs can still maintain hydrogen bond contacts, molecular models suggest that formation of the -GC- staggered cross-link disrupts hydrogen bonding and may also perturb adjacent base pairs leading to an overall reduction in helix stability. Duplexes with specifically positioned and oriented cross-links can be used as substrates to study DNA repair mechanisms.     

M.HhaI binds tightly to substrates containing mismatches at the target base.

The (cytosine-5) DNA methyltransferase M.HhaI causes its target cytosine base to be flipped completely out of the DNA helix upon binding. We have investigated the effects of replacing the target cytosine by other, mismatched bases, including adenine, guanine, thymine and uracil. We find that M.HhaI binds more tightly to such mismatched substrates and can even transfer a methyl group to uracil if a G:U mismatch is present. Other mismatched substrates in which the orphan guanine is changed exhibit similar behavior. Overall, the affinity of DNA binding correlates inversely with the stability of the target base pair, while the nature of the target base appears irrelevant for complex formation. The presence of a cofactor analog. S-adenosyl-L-homocysteine, greatly enhances the selectivity of the methyltransferase for cytosine at the target site. We propose that the DNA methyltransferases have evolved from mismatch binding proteins and that base flipping was, and still is, a key element in many DNA-enzyme interactions.     

HPLC-UV, MALDI-TOF-MS and ESI-MS/MS analysis of the mechlorethamine DNA crosslink at a cytosine-cytosine mismatch pair

Background

Mechlorethamine [ClCH₂CH₂N(CH₃)CH₂CH₂Cl], a nitrogen mustard alkylating agent, has been proven to form a DNA interstrand crosslink at a cytosine-cytosine (C-C) mismatch pair using gel electrophoresis. However, the atomic connectivity of this unusual crosslink is unknown.

Methodology/Principal Findings

HPLC-UV, MALDI-TOF-MS, and ESI-MS/MS were used to determine the atomic connectivity of the DNA C-C crosslink formed by mechlorethamine, MALDI-TOF-MS of the HPLC-purified reaction product of mechlorethamine with the DNA duplex d[CTCACACCGTGGTTC]•d[GAACCACCGTGTGAG] (underlined bases are a C-C mismatch pair) indicated formation of an interstrand crosslink at m/z 9222.088 [M−2H+Na]⁺. Following enzymatic digestion of the crosslinked duplex by snake venom phosphodiesterase and calf intestinal phosphatase, ESI-MS/MS indicated the presence of dC-mech-dC [mech = CH₂CH₂N(CH₃)CH₂CH₂] at m/z 269.2 [M]²⁺ (expected m/z 269.6, exact mass 539.27) and its hydrolytic product dC-mech-OH at m/z 329.6 [M]⁺ (expected m/z 329.2). Fragmentation of dC-mech-dC gave product ions at m/z 294.3 and 236.9 [M]⁺, which are both due to loss of the 4-amino group of cytosine (as ammonia), in addition to dC and dC+HN(CH₃)CH = CH₂, respectively. The presence of m/z 269.2 [M]²⁺ and loss of ammonia exclude crosslink formation at cytosine N⁴ or O² and indicate crosslinking through cytosine N³ with formation of two quaternary ammonium ions.

Conclusions

Our results provide an important addition to the literature, as the first example of the use of HPLC and MS for analysis of a DNA adduct at the N³ position of cytosine.     

Solution structure and stability of the DNA undecamer duplexes containing oxanine mismatch

Solution structures of DNA duplexes containing oxanine (Oxa, O) opposite a cytosine (O:C duplex) and opposite a thymine (O:T duplex) have been solved by the combined use of (1)H NMR and restrained molecular dynamics calculation. One mismatch pair was introduced into the center of the 11-mer duplex of [d(GTGACO(6)CACTG)/d(CAGTGX(17)GTCAC), X = C or T]. (1)H NMR chemical shifts and nuclear Overhauser enhancement (NOE) intensities indicate that both the duplexes adopt an overall right-handed B-type conformation. Exchangeable resonances of C(17) 4-amino proton of the O:C duplex and of T(17) imino proton of O:T duplex showed unusual chemical shifts, and disappeared with temperature increasing up to 30 °C, although the melting temperatures were >50 °C. The O:C mismatch takes a wobble geometry with positive shear parameter where the Oxa ring shifted toward the major groove and the paired C(17) toward the minor groove, while, in the O:T mismatch pair with the negative shear, the Oxa ring slightly shifted toward the minor groove and the paired T(17) toward the major groove. The Oxa mismatch pairs can be wobbled largely because of no hydrogen bond to the O1 position of the Oxa base, and may occupy positions in the strands that optimize the stacking with adjacent bases.     

Structure, dynamics, and thermodynamics of mismatched DNA oligonucleotide duplexes d(CCCAGGG)2 and d(CCCTGGG)2

The structures and hydrogen exchange properties of the mismatched DNA oligonucleotide duplexes d(CCCAGGG)2 and d(CCCTGGG)2 have been studied by high-resolution nuclear magnetic resonance. Both the adenine-adenine and thymine-thymine mismatches are intercalated in the duplexes. The structures of these self-complementary duplexes are symmetric, with the two strands in equivalent positions. The evidence indicates that these mismatches are not stably hydrogen bonded. The mismatched bases in both duplexes are in the anti conformation. The mismatched thymine nucleotide in d(CCCTGGG)2 is intercalated in the duplex with very little distortion of the bases or sugar-phosphate backbone. In contrast, the bases of the adenine-adenine mismatch in d(CCCAGGG)2 must tilt and push apart to reduce the overlap of the amino groups. The thermodynamic data show that the T-T mismatch is less destabilizing than the A-A mismatch when flanked by C-G base pairs in this sequence, in contrast to their approximately equal stabilities when flanked by A-T base pairs in the sequence d(CAAAXAAAG.CTTTYTTTG) where X and Y = A, C, G, and T [Aboul-ela, F., Koh, D., & Tinoco, I., Jr. (1985) Nucleic Acids Res. 13, 4811]. Although the mechanism cannot be determined conclusively from the limited data obtained, exchange of the imino protons with solvent in these destabilized heteroduplexes appears to occur by a cooperative mechanism in which half the helix dissociates.     

Solution structure of an O6-[4-oxo-4-(3-pyridyl)butyl]guanine adduct in an 11 mer DNA duplex: evidence for formation of a base triplex

The pyridyloxobutylating agents derived from metabolically activated tobacco-specific nitrosamines can covalently modify guanine bases in DNA at the O(6) position. The adduct formed, O(6)-[4-oxo-4-(3-pyridyl)butyl]guanine ([POB]dG), results in mutations that can lead to tumor formation, posing a significant cancer risk to humans exposed to tobacco smoke. A combined NMR-molecular mechanics computational approach was used to determine the solution structure of the [POB]dG adduct within an 11mer duplex sequence d(CCATAT-[POB]G-GCCC).d(GGGCCATATGG). In agreement with the NMR results, the POB ligand is located in the major groove, centered between the flanking 5'-side dT.dA and the 3'-side dG.dC base pairs and thus in the plane of the modified [POB]dG.dC base pair, which is displaced slightly into the minor groove. The modified base pair in the structure adopts wobble base pairing (hydrogen bonds between [POB]dG(N1) and dC(NH4) amino proton and between [POB]dG(NH2) amino proton and dC(N3)). A hydrogen bond appears to occur between the POB carbonyl oxygen and the partner dC's second amino proton. The modified guanine purine base, partner cytosine pyrimidine base, and POB pyridyl ring form a triplex via this unusual hydrogen-bonding pattern. The phosphodiester backbone twists at the lesion site, accounting for the unusual phosphorus chemical shift differences relative to those for the control DNA duplex. The helical distortions and wobble base pairing induced by the covalent binding of POB to the O(6)-position of dG help explain the significant decrease of 17.6 degrees C in melting temperature of the modified duplex relative to the unmodified control.     

Structural features and hydration of d(C-G-C-G-A-A-T-T-A-G-C-G); a double helix containing two G.A mispairs

Single crystal X-ray diffraction techniques have been used to characterise the molecular structure of the title compound to 2.5A resolution. The structure consists of ten standard Watson-Crick base pairs and two G.A mismatched base pairs. The purine-purine mismatches have guanine in the usual anti orientation with respect to the sugar and adenine in syn orientation. There are two hydrogen bonds formed between the mismatch bases, N-1 and O-6 of guanine with N-7 and N-6 of adenine respectively. The bulky purine-purine mismatches are accommodated with minor perturbation of the sugar-phosphate backbone. There is a slight improvement in base pair overlap at the mismatch sites. Details of the backbone conformation, base stacking interactions and hydration are presented and compared with those of the parent compound d(C-G-C-G-A-A-T-T-C-G-C-G).     

The effect of methylation of the 6 oxygen of guanine on the structure and stability of double helical DNA

The effect of methylation of the O-6 position of guanine in short segments of double helical DNA has been investigated by molecular mechanical simulations on the sequences d(CGCGCG)2, d(CGC[OMG]CG)2, d(CGT[OMG]CG)2, d(CGC[OMC]CG/(CGCGCG), d(CGC[OMG]CG/d(CGTGCG), d(CGCGAATTCGCG)2 and d(CGCGAATTC[OMG]CG)2. Guanines methylated at the O-6 position are found to form hydrogen bonds of roughly equal strength to cytosine and thymine. The optimum structure of these modified base pairs are not dramatically different from normal GC pairs, but both involve some bifurcation of the proton donors of cytosine (4NH2) or thymine (3NH) between the guanine N3 and O6 groups.     

NMR solution structure of a DNA dodecamer containing a transplatin interstrand GN7-CN3 cross-link.

The DNA duplex d(CTCTCG*AGTCTC).d(GAGAC-TC*GAGAG) containing a single trans- diammine-dichloroplatinum(II) interstrand cross-link (where G* and C* represent the platinated bases) has been studied by two-dimensional NMR. All the exchangeable and non-exchangeable proton resonance lines were assigned (except H5'/H5") and the NOE intensities were transformed into distances via the RELAZ program. By combining the NOESY and COSY data (330 constraints) and NMR-constrained molecular mechanics using JUMNA, a solution structure of the cross-linked duplex has been determined. The duplex is distorted over two base pairs on each side of the interstrand cross-link and exhibits a slight bending of its axis ( approximately 20 degrees ) towards the minor groove. The platinated guanine G* adopts a syn conformation. The rotation results in a Hoogsteen-type pairing between the complementary G(6)* and C(19)* residues which is mediated by the platinum moiety and is stabilized by a hydrogen bond between O6(G(6)*) and N4H(C(19)*). The rise between the cross-linked residues and the adjacent residues is increased owing to the interaction between these adjacent residues and the ammine groups of the platinum moiety. These results are discussed in relation to the slow rate of closure of the monofunctional adducts into interstrand cross-links.     

Effects of adduct formation derived from ethylene dibromide on DNA conformation.

Spectral properties of DNA oligomers containing the single modified guanine, S-[2-(N7-guanyl)ethyl]-glutathione, the major adduct derived from 1,2-dibromoethane, were investigated using UV, CD, and NMR. Two palindromic hexamers, d(ATGCAT) and d(ATCGAT), did not form a duplex with guanine bases modified. When the non self-complementary heptamer, d(CATGCCT), was modified at the single guanine, it formed a duplex with its normal complement d(AGGCATG), although the melting temperature was lowered. However, no duplex formation was observed when a non complementary base other than cytosine was placed in d(AGGXATG), suggesting that non Watson-Crick type base pairs are not stabilized by formation of this adduct.     

Structural Features and Hydration of d(C-G-C-G-A-A-T-T-A-G-C-G); a Double Helix Containing Two G.A Mispairs

Abstract

Single crystal X-ray diffraction techniques have been used to characterise the molecular structure of the title compound to 2.5Å resolution. The structure consists of ten standard Watson-Crick base pairs and two G.A mismatched base pairs. The purine-purine mismatches have guanine in the usual anti orientation with respect to the sugar and adenine in syn orientation. There are two hydrogen bonds formed between the mismatch bases, N-l and 0–6 of guanine with N-7 and N-6 of adenine respectively. The bulky purine-purine mismatches are accommodated with minor perturbation of the sugar-phosphate backbone. There is a slight improvement in base pair overlap at the mismatch sites. Details of the backbone conformation, base stacking interactions and hydration are presented and compared with those of the parent compound d(C-G-C-G-A-A-T-T-C-G-C-G).     

Spermine inhibition of the 2,5-diaziridinyl-1,4-benzoquinone (DZQ) crosslinking reaction with DNA duplexes containing poly(purine). poly(pyrimidine) tracts.

Upon reduction, 2,5-diaziridinyl-1,4-benzoquinone (DZQ) can form an interstrand guanine to guanine crosslink with DNA duplexes containing a d(GC).d(GC) dinucleotide step. The reaction is enhanced by a thymine positioned 5[prime] to each guanine [i.e. in a d(TGCA). d(TGCA) duplex fragment]. Here we show that spermine can inhibit DZQ crosslink formation in duplexes of sequence d[C(N6)TGCA(M6)C]. d[G(M[prime]6)TG-CA(N[prime]6)G]. For N6= M6= GGGGGG, N6= M6= a 'random' sequence and N6= GGGGGG and M6= a 'random' sequence, spermine concentrations of 20, 1 and 3 microM, respectively, were required for 50% inhibition of the DZQ crosslink. This suggests that spermine is more strongly bound to the polyguanosine tract than the random sequence, making it less available for crosslink inhibition. When the polyguanosine tract is interrupted by N 7-deazaguanine (D) located three bases, d(CGGGDGGTGCAGGDGGGC), and four bases, d(CG-GDGGGTGCAGGGDGGC), from the d(TGCA).d(TGCA) site, 30 and 3 microM spermine, respectively, were required for 50% crosslink inhibition. We suggest that this difference is due to the relative proximity of the three-guanosine tract to the d(TGCA).d(TGCA) site. We were able to confirm these conclusions with further experiments using duplexes containing three-guanosine and two-guanosine tracts and from computer simulations of the spermine-DNA complexes.     

Extrahelical cytosine bases in DNA duplexes containing d[GCC]n·d[GCC]n repeats: detection by a mechlorethamine crosslinking reaction

The cytosine–cytosine (C–C) pair is one of the least stable DNA mismatch pairs. The bases of the C–C mismatch are only weakly hydrogen bonded, and previous work has shown that, in certain sequence contexts, they can become unstacked from the core helix, and adopt an ‘extrahelical’ location. Here, using DNA duplexes with d[GCC]_n·d[GCC]_n fragments containing C–C mismatches in a 1,4 bp relationship, we show that cytosine bases of different formal mismatch pairs can be crosslinked by mechlorethamine. For example, in the duplex d[CTCTCGCCGCCGCCGTATC]·d[GATACGCCGCCGCCGAGAG], where underlined cytosine bases are present as the formal C–C mismatch pairs C₇–C₃₂, C₁₀–C₂₉ and C₁₃–C₂₆, we show that two mechlorethamine crosslinks form between C₁₃ and C₂₉ and between C₁₀ and C₃₂, in addition to crosslinks at C₇–C₃₂, C₁₀–C₂₉ and C₁₃–C₂₆ (we have reported previously the crosslinking of formal C–C pairs by mechlorethamine). We interpret the formation of the C₁₃–C₂₉ and C₁₀–C₃₂ crosslinks as evidence of an extrahelical location of the crosslinkable cytosines. Such extrahelical cytosine bases have been observed previously for a single C–C mismatch pair (in the so-called E-motif conformation). In the E-motif, the extrahelical cytosines are folded back towards the 5′-end of the duplex, consistent with our crosslinking data, and also consistent with the absence of C₇–C₂₉ and C₁₀–C₂₆ crosslinks in the current work. Hence, our data provide evidence for an extended E-motif DNA (eE-DNA) conformation in short d[GCC]_n·d[GCC]_n repeat fragments, and raise the possibility that such structures might occur in much longer d[GCC]_n·d[GCC]_n repeat tracts.     

Single base mismatches in DNA. Long- and short-range structure probed by analysis of axis trajectory and local chemical reactivity

We have devised a procedure to generate any single base mismatch in a constant sequence context, and have studied these from two points of view. (1) We have examined electrophoretic mobility of 458 base-pair fragments containing approximately centrally located single mismatches, in polyacrylamide gels, compared to fully matched DNA fragments. We found that no single mismatch caused a significant perturbation of gel mobility, and we conclude that all the mismatches may be accommodated within a helical geometry such that there is no alteration of the path of the helix axis in a straight DNA molecule. (2) We have studied all the single mismatches with respect to reactivity to a number of chemical probes. We found that: (a) No mispaired adenine bases are reactive to diethyl pyrocarbonate and are therefore not simply unpaired such that N-7 is exposed. (b) A number of mispaired thymine bases are reactive to osmium tetroxide, and cytosine bases to hydroxylamine. (c) Where crystal or nuclear magnetic resonance structures are available, the reactivity correlates with exposure of the pyrimidine 5,6 double bonds to attack in the major groove as a result of wobble base-pair formation. This is particularly clear for G.T and I.T base-pairs. (d) Reactivity of bases in mismatched pairs can be dependent on sequence context. (e) Reactivity of the C.C mismatch to hydroxylamine is suppressed at low pH, suggesting that a rearrangement of base-pairing occurs on protonation. The results overall are consistent with the formation of stacked intrahelical base-pairs wherever possible, resulting in no global distortion of the DNA structure, but specific enhancement of chemical reactivity in some cases.     

NMR studies on an oligodeoxynucleotide containing 2-aminopurine opposite adenine

A heteroduplex containing the mismatch 2-aminopurine (AP)-adenine has been synthesized and studied by proton NMR. The mismatch was incorporated into the sequence d[CGG(AP)GGC].d-(GCCACCG). One-dimensional nuclear Overhauser effect measurements in H2O and two-dimensional nuclear Overhauser effect spectra in D2O show AP.A base pairs in a wobble structure in which both bases are in the anti conformation. The adenine is stacked well in the helix, but the helix twist between the adenine and neighboring cytosine in the 3' direction is unusually small. As a result, the aminopurine on the opposite strand is somewhat pushed out of the helix. From the measurements of the imino proton line widths, the two adjacent G.C base pairs are not found to be significantly destabilized by the presence of the purine-purine wobble pair.     

Binding of the antitumor drug nogalamycin and its derivatives to DNA: structural comparison

The three-dimensional molecular structures of the complexes between a novel antitumor drug nogalamycin and its derivative U-58872 with a modified DNA hexamer d[m5CGT(pS)Am5CG] have been determined at 1.7- and 1.8-A resolution, respectively, by X-ray diffraction analyses. Both structures (in space group P6(1)) have been refined with constrained refinement procedure to final R factors of 0.208 (3386 reflections) and 0.196 (2143 reflections). In both complexes, two nogalamycins bind to the DNA hexamer double helix in a 2:1 ratio with the elongated aglycon chromophore intercalated between the CpG steps at both ends of the helix. The aglycon chromophore spans across the GC Watson-Crick base pairs with its nogalose lying in the minor groove and the aminoglucose lying in the major groove of the distorted B-DNA double helix. Most of the sugars remain in the C2'-endo pucker family, except three deoxycytidine residues (terminal C1, C7, and internal C5). All nucleotides are in the anti conformation. Specific hydrogen bonds are found in the complex between the drug and guanine-cytosine bases in both grooves of the helix. One hydroxyl group of the aminoglucose donates a hydrogen bond to the N7 of guanine, while the other receives a hydrogen bond from the N4 amino group of cytosine. The orientation of these two hydrogen bonds suggests that nogalamycin prefers a GC base pair with its aglycon chromophore intercalating at the 5'-side of a guanine (between NpG), or at the 3'-side of a cytosine (between CpN) with the sugars pointing toward the GC base pair. The binding of nogalamycin to DNA requires that the base pairs in DNA open up transiently to allow the bulky sugars to go through, suggesting that nogalamycin prefers GC sequences embedded in a stretch of AT sequences.