Search PubMed⌕ Search

Biomedical subjects

M L Kopka

Publications and source records attributed to M L Kopka.

32 records · Page 2Linked to original sources
PubMed1985

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.

Base Sequence↗
PubMed1985

The molecular origin of DNA-drug specificity in netropsin and distamycin.

X-ray analysis of the complex of netropsin with the B-DNA dodecamer of sequence C-G-C-G-A-A-T-T-BrC-G-C-G reveals that the antitumor antibiotic binds within the minor groove by displacing the water molecules of the spine of hydration. Netropsin amide NH furnish hydrogen bonds to bridge DNA adenine N-3 and thymine O-2 atoms occurring on adjacent base pairs and opposite helix strands, exactly as with the spine of hydration. The narrowness of the groove forces the netropsin molecule to sit symmetrically in the center, with its two pyrrole rings slightly non-coplanar so that each ring is parallel to the walls of its respective region of the groove. Drug binding neither unwinds nor elongates the double helix, but it does force open the minor groove by 0.5-2.0 A, and it bends back the helix axis by 8 degrees across the region of attachment. The netropsin molecule has an intrinsic twist that favors insertion into the minor groove of B-DNA, and it is given a small additional twist upon binding. The base specificity that makes netropsin bind preferentially to runs of four or more A X T base pairs is provided not by hydrogen bonding but by close van der Waals contacts between adenine C-2 hydrogens and CH groups on the pyrrole rings of the drug molecule. Substitution of one or more pyrroles by imidazole could permit recognition of G X C base pairs as well, and it could lead to a class of synthetic "lexitropsins," capable of reading any desired short sequence of DNA base pairs.

Base Sequence↗
PubMed1985

Nuclear Overhauser data and stereochemical considerations suggest that netropsin binds symmetrically within the minor groove of poly(dA).poly(dT), forming hydrogen bonds with both strands of the double helix.

Sarma et al. (J. Biomol. Str. and Dynam. 2, 1085 (1985) have proposed, on the basis of nuclear magnetic resonance experiments on the complex of netropsin with poly(dA).poly(dT), that the drug molecule lies asymmetrically along the dA side of the minor groove and makes hydrogen bonds only with the dA strand. If the crystal structure analyses of B-DNA (Fratini et al., J. Biol. Chem. 257, 14686 (1982] and of its complex with netropsin (Kopka et al., J. Mol. Biol. 183, 553 (1985] are any guide, this off-center, wide-groove model is stereochemically unlikely. More to the point, the off-center model is unnecessary to explain the observed nmr data. All of the nuclear Overhauser and other observations are fully explained by the structure seen in the x-ray crystal analysis, in which netropsin sits squarely centered within the minor groove, making bifurcated hydrogen bonds with both strands.

Binding Sites↗
PubMed1983

Ordered water structure around a B-DNA dodecamer. A quantitative study.

The crystal structure of the double-helical B-DNA dodecamer of sequence C-G-C-G-A-A-T-T-C-G-C-G has been solved and refined independently in three forms: (1) the parent sequence at room temperature; (2) the same sequence at 16 K; and (3) the 9-bromo variant C-G-C-G-A-A-T-TBrC-G-C-G at 7 degrees C in 60% (v/v) 2-methyl-2,4-pentanediol. The latter two structures show extensive hydration along the phosphate backbone, a feature that was invisible in the native structure because of high temperature factors (indicating thermal or static disorder) of the backbone atoms. Sixty-five solvent peaks are associated with the phosphate backbone, or an average of three per phosphate group. Nineteen other molecules form a first shell of hydration to base edge N and O atoms within the major groove, and 36 more are found in upper hydration layers. The latter tend to occur in strings or clusters spanning the major groove from one phosphate group to another. A single spermine molecule also spans the major groove. In the minor groove, the zig-zag spine of hydration that we believe to be principally responsible for stabilizing the B form of DNA is found in all three structures. Upper level hydration in the minor groove is relatively sparse, and consists mainly of strings of water molecules extending across the groove, with few contacts to the spine below. Sugar O-1' atoms are closely associated with water molecules, but these are chiefly molecules in the spine, so the association may reflect the geometry of the minor groove rather than any intrinsic attraction of O-1' atoms for hydration. The phosphate O-3' and O-5' atoms within the backbone chain are least hydrated of all, although no physical or steric impediment seems to exist that would deny access to these oxygen atoms by water molecules.

Chemical Phenomena↗
PubMed1983

A random-walk model for helix bending in B-DNA.

The double-helical B-DNA dodecamer of sequence d(C-G-C-G-A-A-T-T-C-G-C-G) has been refined independently from x-ray crystal structure analyses in five different variants: d(C-G-C-G-A-A-T-T-C-G-C-G) at 16 K, at room temperature, and with bound cis-diamminedichloroplatinum(II), and d(C-G-C-G-A-A-T-T-brC-G-C-G) in 60% 2-methyl-2,4-pentanediol at 20 degrees C and 7 degrees C. These helices display overall axial bends of 22 degrees, 18 degrees, 17 degrees, 14 degrees, and 3 degrees, respectively, providing an opportunity to investigate the nature of the bending process in B-DNA. Bending from one base pair to the next is best described as a stochastic or random-walk process, having forward, retrograde, and sidewise individual steps, but with an overall sense of bending. Individual steps almost always involve rolling of adjacent base pairs over one another along their long axes, not a tilting or wedge displacement that lifts neighboring base pairs apart at one end. A slight preference is observed for bending the double helix in a direction that compresses the major groove rather than the minor, and this is intuitively reasonable in view of the narrowness of the minor groove and its occupation by the spine of hydration that stabilizes the B form of DNA. This model predicts that, when DNA is wound around the nucleosome core, it should not be smoothly curved but should exhibit discrete bends every five base pairs as proposed by Zhurkin et al. [Zhurkin, V.B., Lysov, Y. P. & Ivanov, V. I. (1979) Nucleic Acids Res. 6, 1081-1096)]. Sharper bends may occur at alternate positions, where the major groove faces the nucleosome core.

Base Sequence↗
PubMed1983

A stochastic model for helix bending in B-DNA.

Bending in double-helical B-DNA apparently occurs only by rolling adjacent base pairs over one another along their long axes. The lifting apart of ends that would be required by tilt or wedge angle contributions is too costly in free energy and does not occur. Roll angles at base steps can be positive (compression of major groove) or negative (compression of minor groove), with the former somewhat easier. Individual steps may advance or oppose the overall direction of bend, or make lateral excursions, but the result of this series of "random roll" steps is the production of a net bending in the helix axis. Because the natural roll points for bending in a given plane occur every 5 base pairs, one would expect that double-helical DNA wrapped around a nucleosome core would exhibit bends with the same periodicity. Alternate bends might be particularly acute where the major groove faced the nucleosome core and was compressed against it. The "annealed kinking" model proposed by Fratini et al. (J. Biol. Chem. 257, 14686 (1982) was suggested from the observation that a major bend at a natural roll point is flanked by decreasing roll angles at the steps to either side, as though local strain was being minimized by somewhat blurring the bend out rather than keeping it localized. The random walk model suggested in this paper would describe this as a decreased roll angle as the helix step rotates toward a direction perpendicular to the overall bend. Bending of DNA is seen to be a more stochastic process than had been suspected. Detailed analysis of every helix step reveals both side excursions and backward or retrograde motion, as in any random walk situation. Yet these isolated steps counteract one another, to leave behind a residuum of overall bending in a specific direction.

Base Sequence↗
PubMed1982

Reversible bending and helix geometry in a B-DNA dodecamer: CGCGAATTBrCGCG.

A double-helical B-DNA dodecamer has been analyzed by single crystal x-ray diffraction methods and refined independently in four variants: sequence CGCGAATTCGCG at 20 degrees C and at 16 K, and CGCGAATTBrCGCG in 60% methylpentanediol at 20 and at 7 degrees C. The first three forms show a 14-19 degrees bend in overall helix axis, but the fourth is straight and unbent. Detailed comparisons of the various forms have led to a better understanding of helix geometry and bending. Structural principles can be understood best if organized under four headings: 1) intrinsic geometry of the sugar rings, 2) stacking and relative motion of base pairs, 3) geometry of the connecting phosphate backbone, and 4) mechanics of bending in B-DNA. The observed bending is neither completely localized nor smooth and continuous, but an intermediate compromise that can be termed "annealed kinking."

DNA↗
PubMed1982

The anatomy of A-, B-, and Z-DNA.

Recent advances in DNA synthesis methods have made it possible to carry out single-crystal x-ray analyses of double-stranded DNA molecules of predetermined sequence, with 4 to 12 base pairs. At least one example has been examined from each of the three known families of DNA helix: A, B, and Z. Each family has its own intrinsic restrictions on chain folding and structure. The observed solvent positions in these crystal structures have confirmed earlier fiber and solution measurements, and have led to proposals explaining the transitions from B to A and from B to Z helices. Prospects are improving for an understanding of the mode of bending of DNA in chromatin, and the way in which specific DNA sequences are recognized by drug molecules and repressor proteins.

Crystallography↗
PubMed1980

Large-scale purification of two forms of active lac operator from plasmids.

A practical procedure is described for obtaining milligram quantities of a small (29 nucleotide) Eco RI restriction fragment of DNA containing the Escherichia coli lac operator. A yield of 10--15 mg of operator is obtained from 1 kg of wet cell paste. The resultant operator is shown to be homogeneous and competitively active in filter assays. Two separable but interconvertible forms of lac operator exist in solution, probably linear duplex and hairpin isomers. Only the presumed linear form is active in binding lac repressor by competition assay, but the two isomers are interconvertible by heating to 80 degrees C. The methods described here should be generally applicable for purifying other restriction fragments from plasmids.

Chromatography, Gel↗
PubMed1977

Lac repressor purification without inactivation of DNA binding activity.

A procedure has been developed which eliminates the commonly observed inactivation of the DNA binding activity of the lac repressor during purification. The operator binding activity of the repressor obtained by this method is 100 +/- 10%. The repressor can be stored frozen indefinitely without losing its affinity for DNA.

Coliphages↗