Actinomycin and DNA transcription.

Recent advances in understanding how actinomycin binds to DNA have suggested its mechanism of action. Actinomycin binds to a premelted DNA conformation present within the transcriptional complex. This immobilizes the complex, interfering with the elongation of growing RNA chains. The model has a number of implications for understanding RNA synthesis.

Structure of a novel drug-nucleic acid crystalline complex: 1, 10-phenanthroline-platinum (II) ethylenediamine--5'-phosphoryl-thymidylyl(3'-5') deoxyadenosine.

1,10-Phenanthroline-platinum (II) ethylenediamine (PEPt) forms a 1:2 crystalline complex with 5'-phosphorylthymidylyl (3'-5') deoxyadeno sine (d-pTpA). Crystals are monoclinic, P2, with a = 10.204 A, b = 24.743 A, c = 21.064 A, Beta = 94.6 degrees. The structure has been determined by Patterson and Fourier methods, and refined by least squares to a residual of 0.128 on 2,367 observed reflections. PEPt molecules form sandwich-like stacks with adenine-thymine hydrogen-bonded pairs along the alpha axis. Intercalation in the classic sense is not observed in this structure. Instead, d-pTpA molecules form an open chain structure in which adenine-thymine residues hydrogenbond together with the reversed Hoogsteen type base-pairing configuration. Deoxyadenosine residues exist in the syn conformation and are C3' endo and C1' exo. Thymidine residues are in the high anti conformation with C2' endo puckers. The structure is heavily hydrated, forming a channel-like water network along the alpha axis. Other features of the structure are described.

Reporter molecules as probes of DNA conformation: structure of a crystalline complex containing 2-methyl-4-nitroaniline ethylene dimethylammonium hydrobromide--5-iodocytidylyl (3'-5')guanosine.

2-Methyl-4-nitroaniline ethylene dimethylammonium hydrobromide forms a crystalline complex with the self-complementary dinucleoside monophosphate, 5- iodocytidylyl (3'-5')guanosine. The crystals are tetragonal, with a = b = 32.192 A and c = 23.964 A, space group P4(3)2(1)2. The structure has been solved to atomic resolution by Patterson and Fourier methods, and refined by full matrix least squares. 5- Iodocytidylyl (3'-5')guanosine molecules are held together in pairs through Watson-Crick base-pairing, forming an antiparallel duplex structure. Nitroaniline molecules stack above and below guanine-cytosine pairs in this duplex structure. In addition, a third nitroaniline molecule stacks on one of the other two nitroaniline molecules. The asymmetric unit contains two 5- iodocytidylyl (3'-5')guanosine molecules, three nitroaniline molecules, one bromide ion and thirty-one water molecules, a total of 160 atoms. Details of the structure are described.

A crystalline end product produced by the hydrolytic cleavage of an RNA-like fragment by an organometallointercalator: 1,10-phenanthroline-platinum(II)-ethylenediamine-cytidine 3' monophosphate.

1,10-Phenanthroline-platinum(II)-ethylenediamine ( PEPt ) forms a crystalline complex with cytidine-3'-phosphate (3'-CMP) and its structure has been determined by X-ray crystallography. 3'-CMP molecules are hemiprotonated and form hydrogen-bonded pairs that stack above and below the phenanthroline-platinum(II) drug molecule. Sugar residues are in the C2' endo conformation, with glycosidic torsional angles intermediate between the high and low anti forms. The structure is of particular interest since it forms as an end product of the hydrolytic cleavage of the dinucleoside monophosphate, CpG, by the platinum organometallointercalator ( PEPt ). This hydrolytic activity appears to be specific for the RNA dinucleoside monophosphate fragment, since deoxycytidylyl (3'-5')deoxyguanosine (d-CpG) and other deoxyribooligonucleotides are not cleaved under similar conditions.

Visualization of drug-nucleic acid interactions at atomic resolution. VII. Structure of an ethidium/dinucleoside monophosphate crystalline complex, ethidium: uridylyl(3'-5') adenosine.

Ethidium forms a crystalline complex with the dinucleoside monophosphate, uridylyl (3'-5') adenosine (UpA). The complex crystallizes in the monoclinic space group P2l with unit cell dimensions, a = 13.704 A, b = 31.674 A, c = 15.131 A, beta = 113.9 degrees. This light atom structure has been solved to atomic resolution and refined by full matrix least squares to a residual of 0.12, using 3,034 observed reflections. The asymmetric unit consists of two ethidium molecules, two UpA molecules and 19 solvent molecules, a total of 145 non-hydrogen atoms. The two UpA molecules are hydrogen-bonded together by Watson-Crick type base pairing. Base-pairs in this duplex are separated by 6.7 A; this reflects intercalative binding by one of the ethidium molecules. The other ethidium molecule stacks on either side of the intercalated base-paired dinucleoside monophosphate, being related by a unit cell translation along the a axis. The conformation of the sugar-phosphate backbone accompanying intercalation has been accurately determined in this analysis, and contains the mixed sugar-puckering pattern: C3' endo (3'-5') C2' endo. This same structural feature has been observed in the ethidium-iodoUpA and ethidium-iodoCpG complexes, and exists in two additional structures containing ethidium-CpG. Taken together, these studies confirm our earlier sugar-puckering assignments and demonstrate that iodine covalently bound to the C5 position on uridine or cytosine does not alter the basic sugar-phosphate geometry or the mode of ethidium intercalation in these model studies. We have proposed this stereochemistry to explain the intercalation of ethidium (as well as other simple intercalators) into both DNA and into double-helical RNA, and discuss this aspect of our work further in this paper and in the accompanying papers.

Visualization of drug-nucleic acid interactions at atomic resolution. VIII. Structures of two ethidium/dinucleoside monophosphate crystalline complexes containing ethidium: cytidylyl(3'-5') guanosine.

This paper describes two complexes containing ethidium and the dinucleoside monophosphate, cytidylyl(3'-5')guanosine (CpG). Both crystals are monoclinic, space group P2l, with unit cell dimensions as follows: modification 1: a = 13.64 A, b = 32.16 A, c = 14.93 A, beta = 114.8 degrees and modification 2: a = 13.79 A, b = 31.94 A, c = 15.66 A, beta = 117.5 degrees. Each structure has been solved to atomic resolution and refined by Fourier and least squares methods; the first has been refined to a residual of 0.187 on 1,903 reflections, while the second has been refined to a residual of 0.187 on 1,001 reflections. The asymmetric unit in both structures contains two ethidium molecules and two CpG molecules; the first structure has 30 water molecules (a total of 158 non-hydrogen atoms), while the second structure has 19 water molecules (a total of 147 non-hydrogen atoms). Both structures demonstrate intercalation of ethidium between base-paired CpG dimers. In addition, ethidium molecules stack on either side of the intercalated duplex, being related by a unit cell translation along the a axis. The basic feature of the sugar-phosphate chains accompanying ethidium intercalation in both structures is: C3' endo (3'-5') C2' endo. This mixed sugar-puckering pattern has been observed in all previous studies of ethidium intercalation and is a feature common to other drug-nucleic acid structural studies carried out in our laboratory. We discuss this further in this paper and in the accompanying papers.

Visualization of drug-nucleic acid interactions at atomic resolution. IX. Structures of two N,N-dimethylproflavine: 5-iodocytidylyl (3'-5') guanosine crystalline complexes.

This paper describes two complexes containing N,N-dimethylproflavine and the dinucleoside monophosphate, 5-iodocytidylyl (3'-5') guanosine (iodoCpG). The first complex is triclinic, space group P1, with unit cell dimensions a = 11.78 A, b = 14.55 A, c = 15.50 A, alpha = 89.2 degrees, beta = 86.2 degrees, gamma = 96.4 degrees. The second complex is monoclinic, space group P21, with a = 14.20 A. b = 19.00 A, c = 20.73 A, beta = 103.6 degrees. Both structures have been solved to atomic resolution and refined by Fourier and least squares methods. The first structure has been refined anisotropically to a residual of 0.09 on 5,025 observed reflections using block diagonal least squares, while the second structure has been refined anisotropically to a residual of 0.13 on 2,888 reflections with full matrix least squares. The asymmetric unit in both structures contains two dimethylproflavine molecules and two iodoCpG molecules; the first structure has 16 water molecules (a total of 134 non-hydrogen atoms), while the second structure has 18 water molecules (a total of 136 non-hydrogen atoms). Both structures demonstrate intercalation of dimethylproflavine between base-paired iodoCpG dimers. In addition, dimethylproflavine molecules stack on either side of the intercalated duplex, being related by a unit cell translation along b and a axes, respectively. The basic structural feature of the sugar-phosphate chains accompanying dimethylproflavine intercalation in both structures is the mixed sugar puckering pattern: C3' endo (3'-5') C2' endo. This same structural information is again demonstrated in the accompanying paper, which describes a complex containing dimethylproflavine with deoxyribo-CpG. Similar information has already appeared for other "simple" intercalators such as ethidium, acridine orange, ellipticine, 9-aminoacridine, N-methyl-tetramethylphenanthrolinium and terpyridine platinum. "Complex" intercalators, however, such as proflavine and daunomycin, have given different structural information in model studies. We discuss the possible reasons for these differences in this paper and in the accompanying paper.

Visualization of drug-nucleic acid interactions at atomic resolution. X. Structure of a N,N-dimethylproflavine: deoxycytidylyl(3'-5')deoxyguanosine crystalline complex.

N,N-dimethylproflavine forms a crystalline complex with deoxycytidylyl(3'-5')deoxyguanosine (d-CpG), space group P2(1)2(1)2, with a = 21.37 A, b = 34.05 A, c = 13.63 A. The structure has been solved to atomic resolution and refined by Fourier and least squares methods to a residual of 0.18 on 2,032 observed reflections. The structure consists of two N,N-dimethylproflavine molecules, two deoxycytidylyl (3'-5')deoxyguanosine molecules and 16 water molecules, a total of 128 nonhydrogen atoms. As with other structures of this type, N,N-dimethylproflavine molecules intercalate between base-paired d-CpG dimers. In addition, dimethylproflavine molecules stack on either side of the intercalated duplex, being related by a unit cell translation along the c axis. Both sugar-phosphate chains demonstrate the mixed sugar puckering geometry: C3' endo (3'-5') C2' endo. This same intercalative geometry has been seen in two other complexes containing N,N-dimethylproflavine and iodoCpG, described in the accompanying paper. Taken together, these studies indicate a common intercalative geometry present in both RNA- and DNA- model systems. Again, N,N-dimethylproflavine behaves as a simple intercalator, intercalating asymmetrically between guanine-cytosine base-pairs. The free amino- group on the intercalated dimethylproflavine molecule does not hydrogen bond directly to the phosphate oxygen. Other aspects of the structure will be presented.

Presence of nonlinear excitations in DNA structure and their relationship to DNA premelting and to drug intercalation.

We propose that collectively localized nonlinear excitations (solitons) exist in DNA structure. These arise as a consequence of an intrinsic nonlinear ribose inversion instability that results in a modulated beta alternation in sugar puckering along the polymer backbone. In their bound state, soliton-antisoliton pairs contain beta premelted core regions capable of undergoing breathing motions that facilitate drug intercalation. We call such bound state structures--beta premeltons. The stability of a beta premelton is expected to reflect the collective properties of extended DNA regions and to be sensitive to temperature, pH, ionic strength and other thermodynamic factors. Its tendency to localize at specific nucleotide base sequences may serve to initiate site-specific DNA premelting and melting. We suggest that beta premeltons provide nucleation centers important for RNA polymerase-promoter recognition. Such nucleation centers could also correspond to nuclease hypersensitive sites.

Conformational flexibility in DNA structure and its implications in understanding the organization of DNA in chromatin.

X-ray crystallographic studies of drug-nucleic acid crystalline complexes have suggested that DNA first bends or 'kinks' before accepting an intercalative drug or dye. This flexibility in DNA structure is made possible by altering the normal C2' endo deoxyribose sugar puckering in B DNA to a mixed sugar puckering pattern of the type C3' endo (3'-5') C2' endo and partially unstacking base pairs. A kinking scheme such as this would require minimal sterochemical rearrangement and would also involve small energies. This has prompted us to ask more generally if a conformational change such as this could be used by proteins in their interactions with DNA. Here we describe an interesting superhelical DNA structure formed by kinking DNA every ten base pairs. This structure may be used in the organization of DNA within the nucleosome structure in chromatin.

Mutagen-nucleic acid intercalative binding: structure of a 9-aminoacridine: 5-iodocytidylyl(3'-5')guanosine crystalline complex.

9-Aminoacridine forms a crystalline complex with the dinucleoside monophosphate, 5-iodocytidylyl(3'-5')guanosine. We have solved the three-dimensional structure of this complex by x-ray crystallography and have observed two distinct intercalative binding modes by this drug to miniature Watson-Crick double helical structures. The first of these involves a pseudosymmetric stacking interaction between 9-aminoacridine molecules and guanine-cytosine base-pairs. This configuration may be used by 9-aminoacridine when intercalating into DNA. The second configuration is an asymmetric interaction, largely governed by stacking forces between acridine and guanine rings. This type of association may play an important role in the mechanism of frameshift mutagenesis.