Netropsin interactions in the minor groove of d(GGCCAATTGG) studied by a combination of resolution enhancement and ab initio calculations

The structure of the complex between the minor groove binder netropsin and d(GGCCAATTGG) was determined via single-crystal X-ray techniques. The structure was refined to completion using refmac5.1.24, resulting in a residual R-factor of 20.0% (including 68 water molecules). Using crystal engineering and cryocooling techniques, the resolution could be enhanced to 1.75 A, resulting in an unambiguous determination of the drug conformation and orientation. As previously noticed, bifurcated hydrogen bonds are formed between the amide nitrogen atoms of the drug and the N3 and O2 atoms of A and T base pairs, respectively, clearly cataloging the structure to class I. As the bulky NH2 group on guanine was believed to prevent binding of the drug in the minor groove, the detailed nature of several of the amidinium and guanidinium end contacts were further investigated by ab initio quantum chemical methods.     

Two 1 : 1 binding modes for distamycin in the minor groove of d(GGCCAATTGG).

Single-crystal X-ray structure determinations of the complex between the minor-groove binder distamycin and d(GGCCAATTGG) reveal two 1 : 1 binding modes which differ in the orientation of the drug molecule in the minor groove. The two crystals were grown from different crystallization conditions and found to diffract to 2.38 and 1.85 A, respectively. The structures were refined to completion using SHELXL-93, resulting in a residual R factor of 20.30% for the 2.38-A resolution structure (including 46 water molecules) and 19.74% for the 1.85-A resolution structure (including 74 water molecules). In both orientations, bifurcated hydrogen bonds are formed between the amide nitrogen atoms of the drug and AT base pairs. With a binding site of at least five base pairs, close contacts between the terminal distamycin atoms and guanine amino groups are inevitable. The detailed nature of several of these interactions was further investigated by ab initio quantum chemical methods.     

Stacking interaction of guanine with netropsin in the minor groove of d(CGTATATACG)2

We present the structure of the decanucleotide d(CGTATATACG) determined by single crystal X-ray diffraction at 1.58 A resolution. A netropsin drug is found in the minor groove with guanine stacked on a pyrrole ring of the drug, a feature described here for the first time. The stacked guanine is an extra-helical base coming from the end of a neighbour oligonucleotide. This observation may open the way to the development of minor groove binding drugs with a higher sequence selectivity. The oligonucleotide is in the B-conformation, but the terminal base-pairs are disrupted: the cytosine residues are disordered while the guanine residues penetrate into the minor groove of neighbouring duplexes. Four hydrated Ni ions with octahedral co-ordination are found associated with the N7 atoms of each guanine. The high affinity of these ions with guanine suggests that they may be used as probes for specific guanine residues.     

Guanine specific binding at a DNA junction formed by d[CG(5-BrU)ACG](2) with a topoisomerase poison in the presence of Co(2+) ions

The structure of the duplex d[CG(5-BrU)ACG](2) bound to 9-bromophenazine-4-carboxamide has been solved through MAD phasing at 2.0 A resolution. It shows an unexpected and previously unreported intercalation cavity stabilized by the drug and novel binding modes of Co(2+) ions at certain guanine N7 sites. For the intercalation cavity the terminal cytosine is rotated to pair with the guanine of a symmetry-related duplex to create a pseudo-Holliday junction geometry, with two such cavities linked through the minor groove interactions of the N2/N3 guanine sites at an angle of 40 degrees, creating a quadruplex-like structure. The mode of binding of the drug is shown to be disordered, with the major conformations showing the side chain bound to the N7 position of adjacent guanines. The other end of the duplex exhibits a terminal base fraying in the presence of Co(2+) ions linking symmetry-related guanines, causing the helices to intertwine through the minor groove. The stabilization of the structure by the intercalating drug shows that this class of compound may bind to DNA junctions as well as duplex DNA or to strand-nicked DNA ('hemi-intercalated'), as in the cleavable complex. This suggests a structural basis for the dual poisoning of topoisomerase I and II enzymes by this family of drugs.     

Synthesis and DNA binding studies of a new asymmetric cyanine dye binding in the minor groove of [poly(dA-dT)]2

A new asymmetric cyanine dye has been synthesised and its interaction with different DNA has been investigated. In this dye, BEBO, the structure of the known intercalating cyanine dye BO has been extended with a benzothiazole substituent. The resulting crescent-shape of the molecule is similar to that of the well-known minor groove binder Hoechst 33258. Indeed, comparative studies of BO illustrate a considerable change in binding mode induced by this structural modification. Linear and circular dichroism studies indicate that BEBO binds in the minor groove to [poly (dA-dT)](2), but that the binding to calf thymus DNA is heterogeneous, although still with a significant contribution of minor groove binding. Similar to other DNA binding asymmetric cyanine dyes, BEBO has a large increase in fluorescence intensity upon binding and a relatively large quantum yield when bound. The minor groove binding of BEBO to [poly (dA-dT)](2) affords roughly a 180-fold increase in intensity, which is larger than to that of the commonly used minor groove binding probes DAPI and Hoechst 33258.     

Effects of compound structure on carbazole dication-DNA complexes: tests of the minor-groove complex models

Carbazole dications have shown excellent activity against opportunistic infections, but they are quite different in structure from previously studied unfused aromatic cations that function by targeting the DNA minor groove. In a previous report [Tanious, F. A., Ding, D., Patrick, D. A., Tidwell, R. R., and Wilson, W. D. (1997) Biochemistry 36, 15315-15325] we showed that, despite their fused ring structure, the carbazoles also bind in A/T sequences of the DNA minor groove and we proposed models for the carbazole-DNA complexes with the carbazole nitrogen facing out of the groove for 3,6 substituted compounds but into the groove in 2,7 carbazoles. To test and refine the models, carbazole-N-methyl substituted derivatives have been synthesized in both the 3,6 and 2,7 series as well as a new 2,6 substituted NH derivative that is intermediate in structure. Footprinting results indicate a broad AT specificity of carbazole binding and a pattern in agreement with a minor groove complex. Surface plasmon resonance biosensor analysis of carbazole binding to an oligomer with an AATT central sequence indicated that the 2,7 NH compound has the largest binding constant. Both the 3,6 NH and NMe compounds bind with similar equilibrium constants that are less than for the 2,7 NH compound. The 2,7 NMe compound has the lowest binding constant of all the carbazoles. Spectroscopic results are also similar for the two 3,6 derivatives but are quite different for the 2,7 NH and NMe carbazole dications. Structural analysis of carbazole complexes with an AATT sequence by 2D NMR methods also supported a minor groove complex of the carbazoles in orientations in agreement with the previously proposed models. From these results, it is clear that the fused ring carbazoles can bind strongly in the DNA minor groove with a broad A/T specificity and that the 2,7 and 3,6 substituted carbazoles bind to the minor groove in opposite orientations.     

Minor groove binder antibody conjugates employing a water soluble beta-glucuronide linker

The minor groove binder beta-glucuronide drug-linker 3 was constructed from amino CBI 1 and determined to be a substrate for Escherichia coli beta-glucuronidase (EC 3.2.1.31), resulting in facile drug release. Compound 3 was conjugated to mAbs cAC10 (anti-CD30) and h1F6 (anti-CD70) to give antibody-drug conjugates (ADCs) with potencies comparable to that of free drug 1. The ADCs were largely monomeric at intermediate loading levels (4-5drug/mAb), in contrast to highly aggregated p-aminobenzylcarbamate dipeptide-based ADCs of 1 previously reported. Significant levels of immunologic specificity were observed with cAC10-3 by comparing antigen positive versus negative cell lines and binding versus non-binding control ADCs. The water soluble beta-glucuronide linker is stable in plasma and effectively delivers drugs to target cells leading to potent cytotoxic activities.     

Interactions of DNA binding ligands with PNA-DNA hybrids.

The interactions of two representative mixed-sequence (one with an AT-stretch) PNA-DNA duplexes (10 or 15 base-pairs) and a PNA2/DNA triplex with the DNA binding reagents distamycin A, 4',6-diamidino-2-phenylindole (DAPI), ethidium bromide, 8-methoxy-psoralen and the delta and lambda enantiomers of Ru(phen)2-dppz2+ have been investigated using optical spectroscopic methods. The behaviour of these reagents versus two PNA-PNA duplexes has also been investigated. With triple helical poly(dA)/(H-T10-Lys-NH2)2 no significant intercalative binding was detected for any of the DNA intercalators, whereas DAPI, a DNA minor groove binder, was found to exhibit a circular dichroism with a positive sign and amplitude consistent with minor groove binding. Similarly, a PNA-DNA duplex containing a central AATA motif, a typical minor groove binding site for the DNA minor groove binders distamycin A and DAPI, showed binding for both of these drugs, though with strongly reduced affinity. No important interactions were found for any of the ligands with a PNA-DNA duplex consisting of a ten base-pair mixed purine-pyrimidine sequence with only two AT base-pairs in the centre. Nor did any of the ligands show any detectable binding to the PNA-PNA duplexes (one containing an AATT motif). Various PNA derivatives with extentions of the backbone, believed to increase the flexibility of the duplex to opening of an intercalation slot, were tested for intercalation of ethidium bromide or 8-methoxypsoralen into the mixed sequence PNA-DNA duplex, however, without any observation of improved binding. The importance of the ionic contribution of the deoxyribose phosphate backbone, versus interactions with the nucleobases, for drug binding to DNA is discussed in the light of these findings.     

Effect of structural factors on the stability of duplexes formed by oligonucleotide conjugates with minor groove ligands

The effect of structural factors on the stability of duplexes formed by DNA minor groove binders conjugated with oligonucleotide mono- or diphosphoramidates of the general formula Oligo-MGBm (where Oligo is an oligonucleotide; m = 1 or 2; MGB is -L(Py)2R, L(Py)4R, -L(Im)4R, or -L(Py)4NH(CH2)3CO(Py)4R; Py is a 4-aminopyrrol-2-carboxylic acid residue, L is a gamma-aminobutyric acid or an epsilon-aminocaproic acid residue, R = OEt, NH(CH2)6NEt2, or NH(CH2)6N+Me3) was studied by the method of thermal denaturation. The mode of binder interaction with minor groove depends on the conjugate structure; it may be of the parallel head to head type for bisphosphoramidates and of the antiparallel head to tail type for monophosphoramidates of a hair-pin structure. The effects of the duplexes with parallel orientation (bisphosphoramidates, MGB is L(Py)4R, m = 2) and those of the hairpin structure with the antiparallel orientation (monophosphoramidates, MGB is L(Py)4(CH2)3CO(Py)4R, m = 1) on Tm values were close. The influence of the linker (L) and substituent (R) structures upon Tm was more pronounced for monophosphoramidate (MGB is L(Py)nR, m = 1) than for bisphosphoramidate (MGB is L(Py)nR, m = 2). No more than two oligopyrrolcarboxamide residues (either in parallel or antiparallel orientations) can be incorporated into the duplex minor groove. Moreover, it was shown by the example of monophosphoramidates (Oligo-L(Py)4R and Oligo-L(Py)4NH(CH2)3CO(Py)4R) that the addition of a second ligand capable of incorporation into the minor groove increased Tm of the corresponding duplex in comparison with the duplex formed by the starting monophosphoramidate. At the same time, the introduction of the ligand incapable of incorporating decreased the Tm value. The mode of interaction of the conjugated ligand with the oligonucleotide duplex is determined by its structure. For example, dipyrrolcarboxamide containing an ethoxy group at the ligand C-end stabilizes the duplex due to the stacking interaction with the terminal A*T pair, whereas tetrapyrrolcarboxamides stabilize the duplex by incorporation into the minor groove.     

Use of monoacetyl-4-hydroxyaminoquinoline 1-oxide to probe contacts between guanines and protein in the minor and major grooves of DNA. Interaction of Escherichia coli integration host factor with its recognition site in the early promoter and transposition enhancer of bacteriophage Mu.

Monoacetyl-4-hydroxyaminoquinoline 1-oxide (Ac-HAQO) reacts with DNA to form adducts at the C8- and N2-positions of guanine and with the N6-position of adenine. Only the N2-guanine adduct blocks the 3'-5' exonuclease action of phage T4 DNA polymerase. Piperidine treatment cleaves the DNA at sites bearing C8-guanine adducts. The N2-position of guanine lies in the minor groove of DNA, whereas the C8-position of guanine occupies the major groove. We have taken advantage of these characteristics to employ Ac-HAQO in conjunction with either T4 DNA polymerase or piperidine in a footprinting technique to probe the interaction of the Escherichia coli integration host factor (IHF) with its binding site. We show that when IHF binds to its recognition site both the N2- and C8-positions of guanines are protected from modification by AcHAQO. In addition, the binding of IHF to DNA was prevented when either an N2- or a C8-AQO adduct was present in the binding site. When dimethylsulfate was used as the footprinting reagent, IHF protected against methylation of the N3 position of adenine in the minor groove but not the N7 position of guanine in the major groove. The difference in results obtained with the two reagents is ascribed to their relative sizes. Both DMS and AcHAQO are excluded by IHF from the minor groove, but only the larger AcHAQO molecule is excluded from the major groove.     

Extra-helical guanine interactions in DNA

We review the extra-helical guanine interactions present in many oligonucleotide crystals. Very often terminal guanines interact with other guanines in the minor groove of neighboring oligonucleotides through N2 x N3 hydrogen bonds. In other cases the interaction occurs with the help of Ni2+ ions. Guanine/netropsin stacking in the minor groove has also been found. From these studies we conclude that guanine may have multiple extra-helical interactions. In particular it may be considered a very effective minor groove binder, which could be used in the design of sequence selective binding drugs. Interactions through the major groove are seldom encountered, but might be present when DNA is stretched. Such interactions are also analyzed, since they might be important for homologous chromosome pairing during meiosis.     

Structure refinement of the chromomycin dimer-DNA oligomer complex in solution.

We have refined the initial docking model of the Mg(II)-co-ordinated chromomycin-d(A2G2C2T2) complex (2 drug equivalents per duplex) by a complete relaxation matrix analysis simulation of the two-dimensional nuclear Overhauser effect (NOESY) spectrum of the complex in 2H2O solution. This relaxation matrix refined structure of the complex exhibits the following characteristics. (1) We observe an unwound and elongated duplex that exhibits characteristics distinct from the A and B-DNA family of helices at the central (G-G-C-C).(G-G-C-C) chromomycin dimer binding and flanking sites. On the other hand sugar puckers, glycosidic torsion angles, displacement of the base-pairs from the helix axis and the minor groove width for this central tetranucleotide segment all fall within the A-family of helical parameters. (2) The chromomycin monomers are aligned in a head-to-tail orientation in the Mg(II)-co-ordinated dimer in the complex. The chromophores are aligned with a slight tilt relative to each other and make an angle of 75 degrees between their planes. The C-D-E trisaccharide segments from individual monomers adopt an extended conformation that projects in opposite directions in the dimer. The divalent metal cation is co-ordinated to the O(1) carbonyl and O(9) enolate atoms of the chromophores and aligns them such that the O(9)-Mg-O(9) angle is 170 degrees while all other O-Mg-O angles are in the 95(+/- 15)degrees range. (3) The sequence specificity of the chromomycin dimer for the widened and shallower (G3-G4-C5-C6).(G3-G4-C5-C6) minor groove binding site is associated with intermolecular hydrogen bonds formed between the OH group at C(8) of the chromophore and the minor groove NH2 group at position 2 and N(3) groups of G4 and between the O(1) oxygen of the E-sugar and the minor groove NH2 group at position 2 of G3 in the complex. (4) Additional intermolecular interactions are primarily van der Waals contacts between anomeric and adjacent CH2 protons on each sugar in the C-D-E trisaccharide segments of the chromomycin dimer and the minor groove surface of the DNA. These results provide insights into the induced conformational transitions required to generate a complementary match between the drug dimer and its DNA binding site on complex formation.     

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.     

DNA-sequence specific recognition by a thiazole analogue of netropsin: a comparative footprinting study.

Four different footprinting techniques have been used to probe the DNA sequence selectivity of Thia-Net, a bis-cationic analogue of the minor groove binder netropsin in which the N-methylpyrrole moieties are replaced by thiazole groups. In Thia-Net the ring nitrogen atoms are directed into the minor groove where they could accept hydrogen bonds from the exocyclic 2-amino group of guanine. Three nucleases (DNAase I, DNAase II, and micrococcal nuclease) were employed to detect binding sites on the 160bp tyr T fragment obtained from plasmid pKM delta-98, and further experiments were performed with 117mer and 253mer fragments cut out of the plasmid pBS. MPE.Fe(II) was used to footprint binding sites on an EcoRI/HindIII fragment from pBR322. Thia-Net binds to sites in the minor groove containing 4 or 5 base pairs which are predominantly composed of alternating A and T residues, but with significant acceptance of intrusive GC base pairs. Unlike the parent antibiotic netropsin, Thia-Net discriminates against homooligomeric runs of A and T. The evident preference of Thia-Net for AT-rich sites, despite its containing thiazole nitrogens capable of accepting GC sites by hydrogen bonding, supports the view that the biscationic nature of the ligand imposes a bias due to the electrostatic potential differences in the receptor which favour the ligand reading alternating AT sequences.     

Interactions between an anthracycline antibiotic and DNA: molecular structure of daunomycin complexed to d(CpGpTpApCpG) at 1.2-A resolution

The crystal structure of a daunomycin-d(CGTACG) complex has been solved by X-ray diffraction analysis and refined to a final R factor of 0.175 at 1.2-A resolution. The crystals are in a tetragonal crystal system with space group P4(1)2(1)2 and cell dimensions of a = b = 27.86 A and c = 52.72 A. The self-complementary DNA forms a six base pair right-handed double helix with two daunomycin molecules intercalated in the d(CpG) sequences at either end of the helix. Daunomycin in the complex has a conformation different from that of daunomycin alone. The daunomycin aglycon chromophore is oriented at right angles to the long dimension of the DNA base pairs, and the cyclohexene ring A rests in the minor groove of the double helix. Substituents on this ring have hydrogen-bonding interactions to the base pairs above and below the intercalation site. O9 hydroxyl group of the daunomycin forms two hydrogen bonds with N3 and N2 of an adjacent guanine base. Two bridging water molecules between the drug and DNA stabilize the complex in the minor groove. In the major groove, a hydrated sodium ion is coordinated to N7 of the terminal guanine and the O4 and O5 of daunomycin with a distorted octahedral geometry. The amino sugar lies in the minor groove without bonding to the DNA. The DNA double helix is distorted with an asymmetrical rearrangement of the backbone conformation surrounding the intercalator drug. The sugar puckers are C1,C2'-endo, G2,C1'-endo, C11,C1'-endo, and G12,C3'-exo. Only the C1 residue has a normal anti-glycosyl torsion angle (chi = -154 degrees), while the other three residues are all in the high anti range (average chi = -86 degrees). This structure allows us to identify three principal functional components of anthracycline antibiotics: the intercalator (rings B-D), the anchoring functions associated with ring A, and the amino sugar. The structure-function relationships of daunomycin binding to DNA as well as other related anticancer drugs are discussed.     

Modelling basic features of specificity in DNA-aureolic acid-derived antibiotic interactions

The nonintercalative groove binding of a simplified model of olivomycin, to sequences d(CGCGCGC)2, d(TATATAT)2, and d(CICICIC)2 is investigated. A significant preference is displayed for the minor groove of the d(CG) sequence. This is due predominantly to the formation of H-bonds between the hydroxyl groups on the aglycone of the drug and the 2-amino group of the central guanine of the oligonucleotide.     

Structure and thermodynamics of metal binding in the P5 helix of a group I intron ribozyme.

The solution structure of an RNA hairpin modelling the P5 helix of a group I intron, complexed with Co(NH3)63+, has been determined by nuclear magnetic resonance. Co(NH3)63+, which possesses a geometry very close to Mg(H2O)62+, was used to identify and characterize a Mg2+binding site in the RNA. Strong and positive intermolecular nuclear Overhauser effect (NOE) cross-peaks define a specific complex in which the Co(NH3)63+molecule is in the major groove of tandem G.U base-pairs. The structure of the RNA is characterized by a very low twist angle between the two G.U base-pairs, providing a flat and narrowed major groove. The Co(NH3)63+, although highly localized, is free to rotate to hydrogen bond in several ways to the O4 atoms of the uracil bases and to N7 and O6 of the guanine bases. Negative and small NOE cross-peaks to other protons in the sequence reveal a non-specific or delocalized interaction, characterized by a high mobility of the cobalt ion. Mn2+titrations of P5 show specific broadening of protons of the G.U base-pairs that form the metal ion binding site, in agreement with the NOE data from Co(NH3)63+. Binding constants for the interaction of Co(NH3)63+and of Mg2+to P5 were determined by monitoring imino proton chemical shifts during titration of the RNA with the metal ions. Dissociation constants are on the order of 0.1 mM for Co(NH3)63+and 1 mM for Mg2+. Binding studies were done on mutants with sequences corresponding to the three orientations of tandem G.U base-pairs. The affinities of Co(NH3)63+and Mg2+for the tandem G.U base-pairs depend strongly on their sequences; the differences can be understood in terms of the different structures of the corresponding metal ion-RNA complexes. Substitution of G.C or A.U for G.U pairs also affected the binding, as expected. These structural and thermodynamic results provide systematic new information about major groove metal ion binding in RNA.     

Binding of an antitumor drug to DNA, Netropsin and C-G-C-G-A-A-T-T-BrC-G-C-G

The antitumor antibiotic netropsin has been co-crystallized with a double-helical B-DNA dodecanucleotide of sequence: C-G-C-G-A-A-T-T-BrC-G-C-G, and the structure of the complex has been solved by X-ray diffraction at a resolution of 2.2 A. The structure has been refined independently by Jack-Levitt and Hendrickson-Konnert least-squares methods, leading to a final residual error of 0.257 by the Jack-Levitt approach (0.211 for two-sigma data) or 0.248 by the Hendrickson-Konnert approach, with no significant difference between refined structures. The netropsin molecule displaces the spine of hydration and fits snugly within the minor groove in the A-A-T-T center. It widens the groove slightly and bends the helix axis back by 8 degrees, but neither unwinds nor elongates the double helix. The drug molecule is held in place by amide NH hydrogen bonds that bridge adenine N-3 and thymine O-2 atoms, exactly as with the spine of hydration. The requirement of A X T base-pairs in the binding site arises because the N-2 amino group of guanine would demand impermissibly close contacts with netropsin. It is proposed that substitution of imidazole for pyrrole in netropsin should create a family of "lexitropsins" capable of reading G X C-containing base sequences.     

Interactions of the human T-cell leukemia virus type-II integrase with the conserved CA in the retroviral long terminal repeat end

Retroviral integrases (INs) interact with termini of retroviral DNA in the conserved 5'-C(A/G)T. For most integrases, modifications of critical moieties in the major and minor grooves of these sequences decrease 3'-processing. However, for human immunodeficiency virus type-2 (HTLV-2) IN, the replacement of the guanine with 6-methylguanine or hypoxanthine not only reduced 3'-processing, but also promoted cleavage at a second site. This novel cleavage activity required an upstream ACA, unique to the HTLV-2 U5 end. 3'-Processing assays with additional isosteric modifications at Gua and filter binding experiments revealed that the mechanism of the second site cleavage differed among the major groove, minor groove, and mismatch modifications. Importantly, the decrease in 3'-processing activity noted with the minor groove and mismatch modifications were attributed to a decrease in binding. Major groove modifications, however, decreased the level of 3'-processing, but did not affect binding. This suggests that integrase binds the viral end through the minor groove, but relies on major groove contacts for 3'-processing. Several modifications were also examined in strand transfer and disintegration substrates. HTLV-2 IN showed reduced activity with strand transfer and disintegration substrates containing major groove, but not minor groove modifications. This suggests major groove interactions at guanine also provide an important role in these reactions.     

Crystal and molecular structure of the ammonium salt of the dinucleoside monophosphate d(CpG)

The crystal and molecular structure of the ammonium salt of deoxycytidylyl-(3'-5')-deoxyguanosine has been determined from 0.85 A resolution single crystal X-ray diffraction data. The crystals obtained by acetone diffusion technique at -20 degrees C, are orthorhombic, P212121, a = 12.880(2), b = 17444(2) and c = 27.642(2) A. The structure was solved by high resolution Patterson and Fourier methods and refined to R = 0.136. There are two d(CpG) molecules in the asymmetric unit forming a mini left handed Z-DNA helix. This is in contrast to the earlier reported forms of d(CpG) where the molecules form self base paired duplexes. There are two ammonium ions in the asymmetric unit. The major groove NH+4 ion interacts with N7 of guanines through water bridges besides making H-bonded interactions directly with the phosphate oxygen atoms. A second NH+4 ion is found in the minor groove interacting directly with the phosphate oxygen atoms. Symmetry related molecules pack in such a way that the cytosine base stacks on cytosine and guanine base on guanine. Our structure demonstrates that alternating d(CpG) sequences have the ability to adopt the left handed Z-DNA structure even at the dimer level i.e., in a sequence which is only two base pairs long.