The antitumor agent mitoxantrone binds cooperatively to DNA: evidence for heterogeneity in DNA conformation

The equilibrium binding of the antitumor compound DHAQ, or mitoxantrone [1,4-dihydroxy-5,8-bis[[2-[(2-hydroxyethyl)amino]ethyl]amino]-9,10- anthracenedione], to various DNAs has been examined by optical titration and equilibrium dialysis methods. At low r (bound drug/DNA base pair) values, r less than 0.03, DHAQ binds, in a highly cooperative manner, to calf thymus and Micrococcus lysodeikticus DNAs. The binding isotherms for the interaction of DHAQ with Clostridium perfringens DNA and poly(dA-dT).poly(dA-dT) exhibit a small positive slope at low r values, suggestive of cooperative binding. In contrast, the binding of DHAQ to poly(dG-dC).poly(dG-dC) shows no evidence of cooperative binding even at very low r values. At higher r values (r greater than 0.05), the binding of DHAQ to all the DNAs studied is characterized by a neighbor-exclusion process. A model is proposed to account for the two modes of binding exhibited in the cooperative binding isotherms. The main feature of the proposed model is that local sequence and structural heterogeneity of the DNA give rise to sets of binding sites to which DHAQ binds in a highly cooperative manner, while the majority of the DNA sites bind DHAQ via a neighbor-exclusion process. This two-site model reproduces the observed binding isotherms and leads to the conclusion that DHAQ binds in clusters to selected regions of DNA. It is suggested that clustering may play a role in the physiological activity of drugs.     

N-acetoxy-2-acetylaminofluorene modification of a deoxyoligonucleotide duplex.

The carcinogen N-acetoxy-2-acetylaminofluorene was reacted with d (CCACGCACC) to form a covalent adduct with attachment at the single guanine. The sample was purified, mixed 1:1 with d (GGTGCGTGG) and studied by thermal denaturation experiments. The Tm for the mixture was 35 +/- 3 degrees C, consistent with duplex formation. The method of continuous variation shows that the modified oligomer, d (CCACGAAFCACC), forms a 1:1 duplex with d (GGTGCGTGG). Circular dichroism spectra also indicate the formation of a duplex and suggest that the modified duplex has a left-handed conformation. Addition of the intercalating drug ethidium alters the CD spectrum of the modified duplex, resulting in a CD spectrum similar to that of ethidium bound to right-handed DNA.     

Sequence variation at the major histocompatibility complex locus DQ beta in beluga whales (Delphinapterus leucas) [published erratum appears in Mol Biol Evol 1995 Nov;12(6):1174]

Genetic variation at the Major Histocompatibility Complex locus DQ beta was analyzed in 233 beluga whales (Delphinapterus leucas) from seven populations: St. Lawrence Estuary, eastern Beaufort Sea, eastern Chukchi Sea, western Hudson Bay, eastern Hudson Bay, southeastern Baffin Island, and High Arctic and in 12 narwhals (Monodon monoceros) sympatric with the High Arctic beluga population. Variation was assessed by amplification of the exon coding for the peptide binding region via the polymerase chain reaction, followed by either cloning and DNA sequencing or single-stranded conformation polymorphism analysis. Five alleles were found across the beluga populations and one in the narwhal. Pairwise comparisons of these alleles showed a 5:1 ratio of nonsynonymous to synonymous substitutions per site leading to eight amino acid differences, five of which were nonconservative substitutions, centered around positions previously shown to be important for peptide binding. Although the amount of allelic variation is low when compared with terrestrial mammals, the nature of the substitutions in the peptide binding sites indicates an important role for the DQ beta locus in the cellular immune response of beluga whales. Comparisons of allele frequencies among populations show the High Arctic population to be different (P < or = .005) from the other beluga populations surveyed. In these other populations an allele, Dele-DQ beta*0101-2, was found in 98% of the animals, while in the High Arctic it was found in only 52% of the animals. Two other alleles were found at high frequencies in the High Arctic population, one being very similar to the single allele found in narwhal.      

Sheared Aanti.Aanti base pairs in a destabilizing 2 x 2 internal loop: the NMR structure of 5'(rGGCAAGCCU)2

The 5'(rGGCAAGCCU)(2) duplex contains tandem A.A pairs. The three-dimensional structure of the 5'(rGGCAAGCCU)(2) duplex was modeled by molecular dynamics and energy minimization with NMR-derived distance and dihedral angle restraints. Although the 5'(rCAAG)(2) loop is thermodynamically destabilizing by 1.1 kcal/mol, the tandem A.A pairs adopt a predominant conformation: a sheared anti-anti (A.A trans Hoogsteen/Sugar-edge) alignment similar to that observed in the crystal structure of the P4-P6 domain of the Tetrahymena thermophila intron [Cate, J. H., Gooding, A. R., Podell, E., Zhou, K., Golden, B. L., Kundrot, C. E., Cech, T. R., and Doudna, J. A. (1996) Science 273, 1678-1685]. The NMR-derived structure of the 5'(rGGCAAGCCU)(2) duplex exhibits cross-strand hydrogen bonds from N3 of A4 to an amino hydrogen of A5 and from the 2' oxygen of the A4 sugar to the other amino hydrogen of A5. An intrastrand hydrogen bond is formed from the 2' OH hydrogen of A4 to O5' of A5. The cross-strand A5 bases are stacked. The Watson-Crick G-C regions are essentially A-form. The sheared anti-anti (A.A trans Hoogsteen/Sugar-edge) alignment provides potential contact sites for tertiary interactions and, therefore, is a possible target site for therapeutics. Thus, thermodynamically destabilizing internal loops can be preorganized for tertiary interactions or ligand binding.     

An alternating sheared AA pair and elements of stability for a single sheared purine-purine pair flanked by sheared GA pairs in RNA

A previous NMR structure of the duplex 5'GGU GGA GGCU/PCCG AAG CCG5' revealed an unusually stable RNA internal loop with three consecutive sheared GA pairs. Here, we report NMR studies of two duplexes, 5'GGU GGA GGCU/PCCA AAG CCG5' (replacing the UG pair with a UA closing pair) and 5'GGU GAA GGCU/PCCG AAG CCG5' (replacing the middle GA pair with an AA pair). An unusually stable loop with three consecutive sheared GA pairs forms in the duplex 5'GGU GGA GGCU/PCCA AAG CCG5'. The structure contrasts with that reported for this loop in the crystal structure of the large ribosomal subunit of Deinococcus radiodurans [Harms, J., Schluenzen, F., Zarivach, R., Bashan, A., Gat, S., Agmon, I., Bartels, H., Franceschi, F., and Yonath, A. (2001) Cell 107, 679-688]. The middle AA pair in the duplex 5'GGU GAA GGCU/PCCG AAG CCG5' rapidly exchanges orientations, resulting in alternative base stacking and pseudosymmetry with exclusively sheared pairs. The U GAA G/G AAG C internal loop is 2.1 kcal/mol less stable than the U GGA G/G AAG C internal loop at 37 degrees C. Structural, energetic, and dynamic consequences upon functional group substitutions within related 3 x 3 and 3 x 6 internal loops are also reported.     

NMR reveals the absence of hydrogen bonding in adjacent UU and AG mismatches in an isolated internal loop from ribosomal RNA

NMR studies provide insights into structural features of internal loops. These insights can be combined with thermodynamic studies to generate models for predicting structure and energetics. The tandem mismatch internal loop, 5'GUGG3'(3'CUAC5'), has been studied by NMR. The NMR structure reveals an internal loop with no hydrogen bonding between the loop bases and with the G in the AG mismatch flipped out of the helix. The sequence of this internal loop is highly conserved in rRNA. The loop is located in the large ribosomal subunit and is part of a conserved 58-nt fragment that is the binding domain of ribosomal protein L11. Structural comparisons between variants of this internal loop in crystal structures of the 58-nt domain complexed with L11 protein and of the large ribosomal subunit (LSU) suggest that this thermodynamically destabilizing internal loop is partially preorganized for tertiary interactions and for binding L11. A model for predicting the base pairing and free energy of 2 x 2 nucleotide internal loops with a purine-purine mismatch next to a pyrimidine-pyrimidine mismatch is proposed on the basis of the present NMR structure and previously reported thermodynamics.     

Structure of the anthramycin-d(ATGCAT)2 adduct from one- and two-dimensional proton NMR experiments in solution

One- and two-dimensional 400-MHz proton NMR experiments are used to examine the solution structure of the covalent adduct formed by the interaction of anthramycin methyl ether with the self-complementary deoxyoligonucleotide d(ATGCAT)2. The concentration dependence of chemical shifts and nuclear Overhauser enhancement (NOE) experiments are utilized to assign the adenine H2 protons within the minor groove for both free d(ATGCAT)2 and the adduct. These studies demonstrate that one of the four adenine H2 protons is in close proximity to the bound anthramycin and this results in its upfield shift of 0.3 ppm compared to the adenine H2 protons of the free duplex. Effects of the covalent attachment of anthramycin to the d(ATGCAT)2 duplex result in an increased shielding of selected deoxyribose protons located within the minor groove of the adduct, as demonstrated by two-dimensional autocorrelated (COSY) NMR techniques. Interactions between the protons of the covalently attached anthramycin and the d(ATGCAT)2 duplex are determined by utilizing two-dimensional NOE (NOESY) techniques. Analysis of these data reveals NOE cross-peaks between the anthramycin methyl, H6, and H7 protons with specific deoxyoligonucleotide protons within the minor groove, thus allowing the orientation of the drug within the minor groove to be determined. Nonselective inversion recovery (T1) relaxation experiments are used to probe the structural and dynamic properties of the anthramycin-d(ATGCAT)2 adduct. These data suggest that the binding of anthramycin alters the correlation time of the d(ATGCAT)2 duplex and stabilizes both of the internal A X T base pairs with respect to solvent exchange.(ABSTRACT TRUNCATED AT 250 WORDS)     

Significance of nucleotide sequence alignments: a method for random sequence permutation that preserves dinucleotide and codon usage

The similarity of two nucleotide sequences is often expressed in terms of evolutionary distance, a measure of the amount of change needed to transform one sequence into the other. Given two sequences with a small distance between them, can their similarity be explained by their base composition alone? The nucleotide order of these sequences contributes to their similarity if the distance is much smaller than their average permutation distance, which is obtained by calculating the distances for many random permutations of these sequences. To determine whether their similarity can be explained by their dinucleotide and codon usage, random sequences must be chosen from the set of permuted sequences that preserve dinucleotide and codon usage. The problem of choosing random dinucleotide and codon-preserving permutations can be expressed in the language of graph theory as the problem of generating random Eulerian walks on a directed multigraph. An efficient algorithm for generating such walks is described. This algorithm can be used to choose random sequence permutations that preserve (1) dinucleotide usage, (2) dinucleotide and trinucleotide usage, or (3) dinucleotide and codon usage. For example, the similarity of two 60-nucleotide DNA segments from the human beta-1 interferon gene (nucleotides 196-255 and 499-558) is not just the result of their nonrandom dinucleotide and codon usage.