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Biomedical subjects

A Rich

Publications and source records attributed to A Rich.

At least 163 records · Page 9Linked to original sources
PubMed1990

Molecular structure of nicked DNA: a substrate for DNA repair enzymes.

The molecular structure of a nicked dodecamer DNA double helix, made of a ternary system containing d(CGCGAAAACGCG) + d(CGCGTT) + d(TTCGCG) oligonucleotides, has been determined by x-ray diffraction analysis at 3 A resolution. The molecule adopts a B-DNA conformation, not unlike those found in intact dodecamer DNA molecules crystallized in a somewhat different crystal lattice, despite a gap due to the absence of a phosphate group in the molecule. The helix has a distinct narrow minor groove near the center of the molecule at the AAAA region. This suggests that the internal stabilizing forces due to base stacking and hydrogen-bonding interactions are sufficient to overcome the loss of connectivity associated with the disruption of the covalent backbone of DNA.

Base Sequence↗
PubMed1990

Atomic-resolution structure of the cellulose synthase regulator cyclic diguanylic acid.

Cyclic diguanylic acid acts as a regulator for cellulose synthase activity in the bacterium Acetobacter xylinum. We report the x-ray crystal structure of the regulator at atomic resolution. The structure contains two independent molecules that adopt almost identical conformations. The two molecules form self-intercalated units that are stacked on each other. Two different G.G base-pairing modes occur between the stacks. The more stable one has two or possibly three hydrogen bonds between two guanines and is related to the type of hydrogen bonding that is believed to exist between G-rich strands at the ends of chromosomes.

Acetobacter↗
PubMed1989

Drosophila melanogaster strand transferase. A protein that forms heteroduplex DNA in the absence of both ATP and single-strand DNA binding protein.

The purification of a Drosophila strand transfer protein is described, which involves Bio-Rex 70, Superose 6, Mono S, and single-stranded DNA-agarose chromatography. A 105,000-dalton polypeptide copurifies with the strand transfer activity on the last two column steps. The strand transferase carries out strand transfer at an unusually low protein:single-stranded DNA ratio and requires neither a nucleotide cofactor nor exogenous single-strand DNA binding protein to form heteroduplex DNA. Biochemical analysis of the reaction products has established that one strand of the DNA duplex is displaced during the reaction. Several properties, including the kinetics and stoichiometry of strand transfer, differentiate this activity from previously characterized strand transferases.

Adenosine Triphosphate↗
PubMed1989

Binding of a Hoechst dye to d(CGCGATATCGCG) and its influence on the conformation of the DNA fragment.

Hoechst dye 33258 is a planar drug molecule that binds to the minor groove of DNA, especially where there are a number of A.T base pairs. We have solved the structure of the Hoechst dye bound to the DNA dodecamer d(CGCGATATCGCG) at 2.3 A. This structure is compared to that of the same dodecamer with the minor-groove-binding drug netropsin bound to it, as well as to structures that have been solved for this Hoechst dye bound to a DNA dodecamer containing the central four base pairs with the sequence AATT. We find that the position of the Hoechst drug in this dodecamer is quite different from that found in the other dodecamer since it has an opposite orientation compared to the other two structures. The drug covers three of the four A.T base pairs and extends its piperazine ring to the first G.C base pair adjacent to the alternating AT segment. Furthermore, the drug binding has modified the structure of the DNA dodecamer. Other DNA dodecamers with alternating AT sequences show an alternation in the size of the helical twist between the ApT step (small twist) and the TpA step (large twist). In this structure the alternation is reversed with larger twists in the ApT steps than in the TpA step. In addition, there is a rotation of one of the thymine bases in the DNA dodecamer that is associated with hydrogen bonding to the Hoechst drug. This structure illustrates the considerable plasticity found in the DNA molecule when it binds to different planar molecules inserted into the minor groove.

Base Sequence↗
PubMed1989

The molecular structure of the left-handed Z-DNA double helix at 1.0-A atomic resolution. Geometry, conformation, and ionic interactions of d(CGCGCG).

The structure of d(CGCGCG) crystallized in the presence of magnesium and sodium ions alone is compared to that of the spermine form of the molecule. The very high resolution nature of these structure determinations allows the first true examination of an oligonucleotide structure in fine detail. The values of bond distances and angles are compared to those derived from small molecule crystal structures. In addition, the interactions of cations and polyamines with the Z-DNA helix are analyzed. In particular, multiple cationic charges appear to offer enhanced stabilization for the Z-DNA conformation. The location of spermine molecules along the edge of the deep groove and also spanning the entrance to the groove emphasizes the importance of polyamines for stabilizing this left-handed structure. On averaging, we obtained very similar structural parameters for the two different structures with standard deviations generally smaller than the deviations of the crystallographic model from ideal values. This indicates a high degree of accuracy of the two structures, which have been refined using different data and different refinement methods. The derived bond lengths and angles may thus be more representative of this polymeric DNA structure than those derived from mono- and dinucleotide structures at a similar accuracy.

Base Sequence↗
PubMed1989

Molecular structure of an A-DNA decamer d(ACCGGCCGGT).

The molecular structure of the DNA decamer d(ACCGGCCGGT) has been solved and refined by single-crystal X-ray-diffraction analysis at 0.20 nm to a final R-factor of 18.0%. The decamer crystallizes as an A-DNA double helical fragment with unit-cell dimensions of a = b = 3.923 nm and c = 7.80 nm in the space group P6(1)22. The overall conformation of this A-DNA decamer is very similar to that of the fiber model for A-DNA which has a large average base-pair tilt and hence a wide and shallow minor groove. This structure is in contrast to that of several A-DNA octamers in which the molecules all have low base-pair-tilt angles (8-12 degrees) resulting in an appearance intermediate between B-DNA and A-DNA. The average helical parameters of this decamer are typical of A-DNA with 10.9 base pairs/turn of helix, an average helical twist angle of 33.1 degrees, and a base-pair-tilt angle of 18.2 degrees. However, the CpG step in this molecule has a low local-twist angle of 24.5 degrees, similar to that seen in other A-DNA oligomers, and therefore appears to be an intrinsic stacking pattern for this step. The molecules pack in the crystal using a recurring binding motif, namely, the terminal base pair of one helix abuts the surface of the shallow minor groove of another helix. In addition, the GC base pairs have large propeller-twist angles, unlike those found most other A-DNA structures.

Base Sequence↗
PubMed1989

The propeller DNA conformation of poly(dA).poly(dT).

Physical properties of the DNA duplex, poly(dA).poly(dT) differ considerably from the alternating copolymer poly(dAT). A number of molecular models have been used to describe these structures obtained from fiber X-ray diffraction data. The recent solutions of single crystal DNA dodecamer structures with segments of oligo-A.oligo-T have revealed the presence of a high propeller twist in the AT regions which is stabilized by the formation of bifurcated (three-center) hydrogen bonds on the floor of the major groove, involving the N6 amino group of adenine hydrogen bonding to two O4 atoms of adjacent thymine residues on the opposite strand. Here we show that it is possible to incorporate the features of the single crystal analysis, specifically high propeller twist, bifurcated hydrogen bonds, and a narrow minor groove, as well as the close interstrand NMR signal between adenine HC2 and ribose HC1' of the opposite strand, into a model that is fully compatible with the diffraction data obtained from poly(dA).poly(dT).

Chemical Phenomena↗
PubMed1989

Effects of 5-fluorouracil/guanine wobble base pairs in Z-DNA: molecular and crystal structure of d(CGCGFG).

The chemotherapeutic agent 5-fluorouracil is a DNA base analogue which is known to incorporate into DNA in vivo. We have solved the structure of the oligonucleotide d(CGCGFG), where F is 5-fluorouracil (5FU). The DNA hexamer crystallizes in the Z-DNA conformation at two pH values with the 5FU forming a wobble base pair with guanine in both crystal forms. No evidence of the enol or ionized form of 5FU is found under either condition. The crystals diffracted X-rays to a resolution of 1.5 A and their structures have been refined to R-factors of 20.0% and 17.2%, respectively, for the pH = 7.0 and pH = 9.0 forms. By comparing this structure to that of d(CGCGCG) and d(CGCGTG), we were able to demonstrate that the backbone conformation of d(CGCGFG) is similar to that of the archetypal Z-DNA. The two F-G wobble base pairs in the duplex are structurally similar to the T-G base pairs both with respect to the DNA helix itself and its interactions with solvent molecules. In both cases water molecules associated with the wobble base pairs bridge between the bases and stabilize the structure. The fluorine in the 5FU base is hydrophobic and is not hydrogen bonded to any solvent molecules.

Base Composition↗
PubMed1989

Molecular structure of the netropsin-d(CGCGATATCGCG) complex: DNA conformation in an alternating AT segment.

The molecular structure of the complex between a minor groove binding drug (netropsin) and the DNA dodecamer d(CGCGATATCGCG) has been solved and refined by single-crystal X-ray diffraction analysis to a final R factor of 20.0% to 2.4-A resolution. The crystal is similar to that of the other related dodecamers with unit cell dimensions of a = 25.48 A, b = 41.26 A, and c = 66.88 A in the space group P2(1)2(1)2(1). In the complex, netropsin binds to the central ATAT tetranucleotide segment in the narrow minor groove of the dodecamer B-DNA double helix as expected. However, in the structural refinement the drug is found to fit the electron density in two orientations equally well, suggesting the disordered model. This agrees with the results from solution studies (chemical footprinting and NMR) of the interactions between minor groove binding drugs (e.g., netropsin and distamycin A) and DNA. The stabilizing forces between drug and DNA are provided by a combination of ionic, van der Waals, and hydrogen-bonding interactions. No bifurcated hydrogen bond is found between netropsin and DNA in this complex due to the unique dispositions of the hydrogen-bond acceptors (N3 of adenine and O2 of thymine) on the floor of the DNA minor groove. Two of the four AT base pairs in the ATAT stretch have low propeller twist angles, even though the DNA has a narrow minor groove. Alternating helical twist angles are observed in the ATAT stretch with lower twist in the ApT steps than in the TpA step.

Base Composition↗
PubMed1989

Lateralised dual-task performance: the effect of muscular-repositioning.

In two experiments subjects performed tapping tasks requiring different degree of muscular repositioning while concurrently performing a speech task. In all concurrent speech conditions right-hand performance was disrupted more than left-hand performance, but the degree of this differential disruption did not alter as a function of the muscular repositioning requirements of the dual-tasks. It was concluded that rapid muscular repositioning does not appear to be the source of lateralised interference effects observed in concurrent task studies. However, it may be argued that some minimum level of repositioning is required, particularly if the differential effect reflects a lateralisation of "kind" rather than of "degree". This needs further research. One relevant factor proposed here which may alter the degree of lateralised effects is the degree of muscular effort required to perform each of the dual-tasks.

Adult↗
PubMed1989

Polyamine interaction with Z-DNA.

In order to elucidate the detailed Z-DNA interaction with polyamines and also to clarify the mutual molecular recognition between the left-handed helix and the biologically important polyamine molecule, several polyamine-Z-DNA hexamer complexes were crystallized and their crystal structures were determined by X-ray diffraction. The general interaction modes found in these crystal structures were discussed in comparison with those in the complexes between polyamine and the right-handed DNA or RNA.

Crystallization↗
PubMed1989

An estimate of the extent of folding of nucleosomal DNA by laterally asymmetric neutralization of phosphate groups.

We attempt quantitative implementation of a previous suggestion that asymmetric charge neutralization of DNA phosphate groups may provide part of the driving force for nucleosome folding. Polyelectrolyte theory can be used to estimate the effective compressive force acting along the length of one side of the DNA surface when a fraction of the phosphate groups are neutralized by histones bound to that side. A standard engineering formula then relates the force to the bending amplitude caused by it. Calculated bending amplitudes are consistent with the curvature of nucleosomal DNA and the overall extent of charge neutralization by the histones. The relation of the model to various aspects of nucleosome folding, including the detailed path of core-particle DNA, is discussed. Several other DNA-protein complexes are listed as examples of possible asymmetric charge-induced bending.

DNA↗
PubMed1989

The level of Z-DNA in metabolically active, permeabilized mammalian cell nuclei is regulated by torsional strain.

Permeabilized nuclei from mammalian cells encapsulated within agarose microbeads in an isotonic buffer are active in transcription and replication (Jackson, D. A., and P. R. Cook. 1985. EMBO (Eur. Mol. Biol. Organ.) J. 4:913-918). Their DNA is intact and the nuclei are accessible to macromolecules. Myeloma nuclei prepared in this way were used to probe the extent of DNA negative supercoiling and the effects of altering torsional strain by binding radioactively labeled monoclonal antibodies to Z-DNA. Control experiments used monoclonal antibodies against a nonhistone chromosomal protein, HMG-17. On increasing the amount of anti-HMG-17 added, a binding plateau was reached encompassing a 200-fold range of antibody concentration. On binding anti-Z-DNA antibody, a similar broad plateau of constant binding was found encompassing a 100-fold range of antibody concentration. The latter result was taken as a measure of preexisting Z-DNA in the nuclei. Additional anti-Z-DNA antibody binding can be "induced" in the presence of much higher concentration of antibody, apparently by perturbing the B-DNA/Z-DNA equilibrium. On inhibiting topoisomerase I with camptothecin, an elevated antibody binding plateau was found, suggesting that elastic torsional strain in the DNA is responsible for stabilizing the preexisting Z-DNA. This interpretation is supported by the fact that addition of small, nicking amounts of DNase I leads to a complete loss of antibody binding in the Z-DNA plateau region but not in the region of "induced" Z-DNA.

Animals↗
PubMed1989

Nonrandom distribution of long mono- and dinucleotide repeats in Drosophila chromosomes: correlations with dosage compensation, heterochromatin, and recombination.

Long stretches of (dC-dA)n.(dT-dG)n, abbreviated CA/TG, have a distinctive distribution on Drosophila chromosomes (M.L. Pardue, K. Lowenhaupt, A. Rich, and A. Nordheim, EMBO J. 6:1781-1789, 1987). The distribution of CA/TG suggests a correlation with the overall transcriptional activity of chromosomal regions and with the ability to undergo meiotic recombination. These correlations are conserved among Drosophila species and may indicate one or more chromosomal functions. To test the generality of these findings, we analyzed the distribution of the rest of the six possible mono- and dinucleotide repeats (A/T, C/G, AT/AT, CA/TG, CT/AG, and CG/CG). All but CG/CG were present at significant levels in the genomes of the six Drosophila species studied; however, A/T levels were an order of magnitude lower than those of the other sequences. Data base analyses suggested that the same sequences are present in other eucaryotes. Like CA/TG, both CT/AG and C/G showed increased levels on dosage-compensating chromosomes; however, the individual sites clearly differed for each sequence. In contrast, A/T and AT/AT, although present in Drosophila DNA, could not be detected in situ in polytene chromosomes. We also used in situ hybridization to analyze the neo-Y chromosome of Drosophila miranda, an ancestral autosome that has become attached to the Y chromosome and is now partially heterochromatic. The neo-Y has acquired repeated DNA sequences; we found that the added sequences are as devoid of mono- and dinucleotide repeats as other heterochromatin. The distribution and function of these sequences are likely to result from both their repetitious nature and base contents.

Animals↗
PubMed1989

The human recombination strand exchange process.

A mechanism for the initiation of general recombination that involves the formation of left-handed Z-DNA heteroduplex segments adjacent to right-handed B-DNA heteroduplex segments is discussed. The paranemic nature of this initiation structure allows for homology recognition in the absence of strand cleavage. This model suggests that proteins catalyzing recombination initiation via the formation of paranemic joint should in some capacity recognize Z-DNA. Other studies have shown that both the RecA protein of Escherichia coli and the Rec1 protein of Ustilago maydis have a greater affinity for Z-DNA than B-DNA. Here we have used Z-DNA affinity chromatography to purify a peptide of approximately 120 kilodaltons from a human tumor cell line that catalyzes a simple recombination strand-transfer reaction similar to one developed for the characterization of the RecA and Rec1 proteins. We report details of the characterization of the human strand-transfer activity and identified a potential human recombination complex.

Affinity Labels↗