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Biomedical subjects

R E Dickerson

Publications and source records attributed to R E Dickerson.

At least 19 recordsLinked to original sources
PubMed1998

DNA bending: the prevalence of kinkiness and the virtues of normality.

DNA bending in 86 complexes with sequence-specific proteins has been examined using normal vector plots, matrices of normal vector angles between all base pairs in the helix, and one-digit roll/slide/twist tables. FREEHELIX, a new program especially designed to analyze severely bent and kinked duplexes, generates the foregoing quantities plus local roll, tilt, twist, slide, shift and rise parameters that are completely free of any assumptions about an overall helix axis. In nearly every case, bending results from positive roll at pyrimidine-purine base pair steps: C-A (= T-G), T-A, or less frequently C-G, in a direction that compresses the major groove. Normal vector plots reveal three well-defined types of bending among the 86 examples: (i) localized kinks produced by positive roll at one or two discrete base pairs steps, (ii) three-dimensional writhe resulting from positive roll at a series of adjacent base pairs steps, or (iii) continuous curvature produced by alternations of positive and negative roll every 5 bp, with side-to-side zig-zag roll at intermediate position. In no case is tilt a significant component of the bending process. In sequences with two localized kinks, such as CAP and IHF, the dihedral angle formed by the three helix segments is a linear function of the number of base pair steps between kinks: dihedral angle = 36 degrees x kink separation. Twenty-eight of the 86 examples can be described as major bends, and significant elements in the recognition of a given base sequence by protein. But even the minor bends play a role in fine-tuning protein/DNA interactions. Sequence-dependent helix deformability is an important component of protein/DNA recognition, alongside the more generally recognized patterns of hydrogen bonding. The combination of FREEHELIX, normal vector plots, full vector angle matrices, and one-digit roll/slide/twist tables affords a rapid and convenient method for assessing bending in DNA.

Bacterial Proteins↗
PubMed1998

The theoretical limits of DNA sequence discrimination by linked polyamides.

Linked polyamides bind in the minor groove of double-stranded DNA in a partially sequence-specific manner. This report analyzes the theoretical limits of DNA sequence discrimination by linked polyamides composed of two to four different types of heterocyclic rings, determining (i) the optimal choice of base-binding specificity for each ring and (ii) the optimal design for a polyamide composed of these rings to target a given DNA sequence and designed to maximize the fraction of the total polyamide binding to the specified target sequence relative to all other sequences. The results show that, fortuitously, polyamides composed of pyrrole, a naturally occurring G-excluding element, and imidazole, a rationally designed G-favoring element, have features similar to the theoretical optimum design for polyamides composed of two different rings. The results also show that, in polyamides composed of two or three types of heterocyclic rings, choosing a nonspecific "placeholder" ring, which binds equally strongly to each of the four bases, along with one or two base-specific rings will often enhance sequence specificity over a polyamide composed entirely of base-specific rings.

Amides↗
PubMed1998

NarL dimerization? Suggestive evidence from a new crystal form.

The structure of the Escherichia coli response regulator NarL has been solved in a new, monoclinic space group, and compared with the earlier orthorhombic crystal structure. Because the monoclinic crystal has two independent NarL molecules per asymmetric unit, we now have three completely independent snapshots of the NarL molecule: two from the monoclinic form and one from the orthorhombic. Comparison of these three structures shows the following: (a) The pairing of N and C domains of the NarL molecule proposed from the earlier analysis is in fact correct, although the polypeptide chain connecting domains was, and remains, disordered and not completely visible. The new structure exhibits identical relative orientation of N and C domains, and supplies some of the missing residues, leaving a gap of only seven amino acids. (b) Examination of corresponding features in the three independent NarL molecules shows that deformations in structure produced by crystal packing are negligible. (c) The "telephone receiver" model of NarL activation is confirmed. The N domain of NarL blocks the binding of DNA to the C domain that would be expected from the helix-turn-helix structure of the C domain. Hence, binding can only occur after significant displacement of N and C domains. (d) NarL monomers have a strong tendency toward dimerization involving contacts between helixes alpha 1 in the two monomers, and this may have mechanistic significance in DNA binding. Analogous involvement of helix alpha 1 in intermolecular contacts is also found in UhpA and in the CheY/CheZ complex.

Amino Acid Sequence↗
PubMed1997

Defining GC-specificity in the minor groove: side-by-side binding of the di-imidazole lexitropsin to C-A-T-G-G-C-C-A-T-G.

BACKGROUND: Polyamide drugs, such as netropsin, distamycin and their lexitropsin derivatives, can be inserted into a narrow B-DNA minor groove to form 1:1 complexes that can distinguish AT base pairs from GC, but cannot detect end-for-end base-pair reversals such as TA for AT. In contrast, 2:1 side-by-side polyamide drug complexes potentially are capable of such discrimination. Imidazole (Im) and pyrrole (Py) rings side-by-side read a GC base pair with the Im ring recognizing the guanine side. But the reason for this specific G-Im association is unclear because the guanine NH2 group sits in the center of the groove. A 2:1 drug:DNA complex that presents Im at both ends of a GC base pair should help unscramble the issue of imidazole reading specificity. RESULTS: We have determined the crystal structure of a 2:1 complex of a di-imidazole lexitropsin (DIM), an analogue of distamycin, and a DNA decamer with the sequence C-A-T-G-G-C-C-A-T-G. The two DIM molecules sit antiparallel to one another in a broad minor groove, with their cationic tails widely separated. Im rings of one drug molecule stack against amide groups of the other. DIM1 rests against nucleotides C7A8T9G10 of strand 1 of the helix, whereas DIM2 rests against G14G15C16C17 on strand 2. All DIM amide nitrogens donate hydrogen bonds to N and O atoms on the floor of the DNA groove and, in addition, the two Im rings on DIM2 accept hydrogen bonds from guanine N2 amines, thereby providing specific reading. The guanine N2 amine can bond to Im on its own side of the groove, but not on the cytosine side, because of limits on close approach of the two Im rings and the geometry of sp2 hybridization about the amide nitrogen. CONCLUSIONS: Im and Py rings distinguish AT from GC base pairs because of steric factors involving the bulk of the guanine amine, and the ability of Im to form a hydrogen bond with the amine. Side-by-side Im and Py rings differentiate GC from CG base pairs because of tight steric contacts and sp2 hybridization at the amine nitrogen atom, with the favored conformations being G/Im,Py/C and C/Py,Im/G. Discrimination between AT and TA base pairs may be possible using bulkier rings, such as thiazole to select the A end of the base pair.

Binding Sites↗
PubMed1997

Structure of a DNA analog of the primer for HIV-1 RT second strand synthesis.

The non-self-complementary DNA decamer C-A-A-A-G-A-A-A-A-G/C-T-T-T-T-C-T-T-T-G is a DNA/DNA analogue of a portion of the polypurine tract or PPT, which is a RNA/DNA hybrid that serves as a primer for synthesis of the (+) DNA strand by HIV reverse transcriptase (RT), and which is not digested by the RNase H domain of reverse transcriptase following (-) strand synthesis. The same unusual conformation that eludes RNase H, thought to be a change in width of minor groove, may also be responsible for the inhibition of HIV RT by minor groove binding drugs such as distamycin and their bis-linked derivatives. The present X-ray crystal structure of this DNA decamer exhibits the usual properties of A-tract B-DNA under biologically relevant conditions: large propeller twist of base-pairs, narrowed minor groove, and a straight helix axis. Groove narrowing is fully developed in the A-A-A-A region, but not in the A-A-A region, which previous investigators have proposed as being too short to exhibit typical A-tract properties. The RNA/DNA hybrid produced by HIV reverse transcriptase during (-) strand synthesis presumably forms a "heteromerous" or H-helix with narrower minor groove than an A-helical RNA/RNA duplex. If the narrowing of minor groove in A-tract H-helices is comparable to that seen in A-tract B-helices, then the narrowed minor groove of the polypurine tract could make the second primer site both (1) impervious to RNase H digestion, and (2) susceptible to inhibition by minor groove binding drugs.

Crystallography, X-Ray↗
PubMed1997

Estimation of the DNA sequence discriminatory ability of hairpin-linked lexitropsins.

Three- and four-ring polyamides containing N-methylimidazole and N-methylpyrrole, and their hairpin-linked derivatives, bind side-by-side in the minor groove of DNA in a sequence-specific manner. The sequences recognized by side-by-side molecules are dependent on the pairings of the polyamide rings to the bases. In this study we report a mathematical model for estimating the free energies of binding for gamma-aminobutyric acid-linked polyamides to 5- and 6-bp DNA sequences. The model parameters are calibrated by a least-squares fit to 35 experimental binding constants. The model performs well in cross-validation experiments and the parameters are consistent with previously proposed empirical rules of polyamide-DNA binding. We apply the model to the design of targeted polyamides, evaluating the ability of the proposed polyamides to bind to a DNA sequence of interest while minimizing binding to the remaining DNA sequences.

Antineoplastic Agents↗
PubMed1997

Design of stapled DNA-minor-groove-binding molecules with a mutable atom simulated annealing method.

We report the design of optimal linker geometries for the synthesis of stapled DNA-minor-groove-binding molecules. Netropsin, distamycin, and lexitropsins bind side-by-side to mixed-sequence DNA and offer an opportunity for the design of sequence-reading molecules. Stapled molecules, with two molecules covalently linked side-by-side, provide entropic gains and restrain the position of one molecule relative to its neighbor. Using a free-atom simulated annealing technique combined with a discrete mutable atom definition, optimal lengths and atomic composition for covalent linkages are determined, and a novel hydrogen bond 'zipper' is proposed to phase two molecules accurately side-by-side.

Base Sequence↗
PubMed1997

Evaluation of a cassette-screen-film combination for radiation therapy portal localization imaging with improved contrast.

A traditional limitation with radiation therapy portal images is low image contrast, due in part to the low attenuation of the exposing radiation by the tissues being imaged, and the contrast capabilities of the image receptor. We have developed, and have clinically evaluated, a cassette-screen-film combination for portal localization imaging, which features a copper front screen plus Gd2O2S:Tb fluorescent screens and a slow-speed, fine grain, film emulsion with inherently high contrast coated on both sides of a 7 mil Estar base. The film can be processed in a conventional rapid-process film processor. Sensitometric data indicate that the film contrast (average gradient) for the new combination is approximately 3.5 times higher than the conventional portal localization systems in current use. The new combination has been clinically compared with two conventional systems. The required monitor unit settings were found to be similar. Initial clinical results indicate portal images made with the new combination are superior to those obtained with the conventional combinations. The images have much higher contrast, subjective impressions of lower noise, show clearer definition of structures, and are much easier to read.

Biophysical Phenomena↗
PubMed1996

Linked lexitropsins and the in vitro inhibition of HIV-1 reverse transcriptase RNA-directed DNA polymerization: a novel induced-fit of 3,5 m-pyridyl bisdistamycin to enzyme-associated template-primer.

Five classic DNA minor groove-binding drugs and a series of bis-linked lexitropsins based on netropsin and distamycin have been screened for their effectiveness in inhibiting transcription by HIV-1 reverse transcriptase (RT) on a poly(rA).oligo(dT) template-primer (TP). The two most effective drugs, 3,5 m-pyridyl-linked bisdistamycin (MPyr) and trans-vinyl-linked bisdistamycin (TVin), show (1) enhanced inhibition in reactions initiated with pre-incubated enzyme template-primer (ETP) and (2) reduced affinity for a "free" TP analog, when compared with the parent drug distamycin. All three drugs lack the ability to inhibit processive incorporation of nucleotide, suggesting drug intervention instead at initiation or termination of processive cycles. The two bis-linked drugs exhibit different kinetic behavior with reverse transcriptase's two substrates: template-primer and nucleotide. When primer is the variable substrate, TVin is partially noncompetitive and MPyr is dead-end competitive (Ki = 6.5 microM). With nucleotide as substrate, TVin is noncompetitive at low drug concentrations and MPyr is uncompetitive. Gel band mobility shift assays with MPyr indicate that the drug inhibits via entrapment of TP on the enzyme rather than displacement of TP from the enzyme surface. The conformation of nucleic acid is most likely altered upon MPyr binding, enhancing the induced fit of enzyme to hybrid duplex. The relevance of this novel mode of inhibition is considered in relation to enzyme association/dissociation with TP that occurs prior to (-)-DNA strand transfer, and to the structural implications of an enzyme-bound hybrid RNA/DNA nucleic acid.

Antiviral Agents↗
PubMed1996

Structure of the Escherichia coli response regulator NarL.

The crystal structure analysis of the NarL protein provides a first look at interactions between receiver and effector domains of a full-length bacterial response regulator. The N-terminal receiver domain, with 131 amino acids, is folded into a 5-strand beta sheet flanked by 5 alpha helices, as seen in CheY and in the N-terminal domain of NTRC. The C-terminal DNA-binding domain, with 62 amino acids, is a compact bundle of 4 alpha helices, of which the middle 2 form a helix-turn-helix motif closely related to that of Drosophila paired protein and other H-T-H DNA-binding proteins. The 2 domains are connected by an alpha helix of 10 amino acids and a 13-residue flexible tether that is not visible and presumably disordered in the X-ray structure. In this unphosphorylated form of NarL, the C-terminal domain is turned against the receiver domain in a manner that would preclude DNA binding. Activation of NarL via phosphorylation of Asp59 must involve transfer of information to the interdomain interface and either rotation or displacement of the DNA-binding C-terminal domain. Docking of a B-DNA duplex against the isolated C-terminal domain in the manner observed in paired protein and other H-T-H proteins suggests a stereochemical basis for DNA sequence preference: T-R-C-C-Y (high affinity) or T-R-C-T-N (low affinity), which is close to the experimentally observed consensus sequence: T-A-C-Y-N. The NarL structure is a model for other members of the FixJ or LuxR family of bacterial transcriptional activators, and possibly to the more distant OmpR and NtrC families as well.

Amino Acid Sequence↗
PubMed1996

How proteins recognize the TATA box.

The crystal structure of a complex of human TATA-binding protein with TATA-sequence DNA has been solved, complementing earlier TBP/DNA analyses from Saccharomyces cerevisiae and Arabidopsis thaliana. Special insight into TATA box specificity is provided by considering the TBP/DNA complex, not as a protein molecule with bound DNA, but as a DNA duplex with a particularly large minor groove ligand. This point of view provides explanations for: (1) why T.A base-pairs are required rather than C.G; (2) why an alternation of T and A bases is needed; (3) how TBP recognizes the upstream and downstream ends of the TATA box in order to bind properly; and (4) why the second half of the TATA box can be more variable than the first.

Amino Acid Sequence↗
PubMed1996

MPD and DNA bending in crystals and in solution.

Bending of 15 to 24 degrees is observed within crystal structures of B-DNA duplexes, is strongly sequence-dependent, and exhibits no correlation with the concentration of MPD (2-methyl-2,4-pentanediol) in the crystallizing solution. Two types of bends are observed: facultative bends or flexible hinges at junctions between regions of G.C and A.T base-pairs, and a persistent and almost obligatory bend at the center of the sequence R-G-C-Y. Only A-tracts are characteristically straight and unbent in every crystal structure examined to date. A detailed examination of normal vector plots for individual strands of a double helix provides an explanation, in terms of the stacking properties of guanine and adenine bases. The effect of high MPD concentrations, in both solution and crystal, is to decrease local bending somewhat without removing it altogether. MPD gel retardation experiments provide no basis for choosing among the three models that seek to explain macroscopic curvature of DNA by means of microscopic bending: junction being, bent A-tracts, or bent general -sequence DNA. Crystallographic data on the straightness of A-tracts, the bendability of non-A sequences, and the identity of inclination angles in A-tract and non-A-tracts B-DNA support only the general-sequence bending model. The pre-melting transition observed in A-tract DNA probably represents a relaxation of stiff adenine stacks to a flexible conformation more typical of general-sequence DNA.

Adenine↗
PubMed1995

Structure of a dicationic monoimidazole lexitropsin bound to DNA.

An X-ray crystal structure has been solved of the complex of a dicationic lexitropsin with a B-DNA duplex of sequence CGCGAATTCGCG. The lexitropsin is identical to netropsin except for replacement of the first methylpyrrole ring by methylimidazole, converting a =CH- to =N-. Crystals are isomorphous with those of the DNA dodecamer in the absence of drug. Although the =N- for =CH- substitution was intended to make that locus on the drug molecule compatible with a G.C base pair, electrostatic attraction for the two cationic ends of the drug predominates, and this lexitropsin binds to the same central AATT site as does the parent netropsin. But unlike netropsin, this lexitropsin exhibits end-for-end disorder in the crystal. Both orientations were refined separately to completion. Final residual errors at 2.25 A resolution for the 2358 reflections above 2 sigma in F are R = 0.165 for one orientation (LexA) with 37 water molecules and 0.164 for the inverted drug orientation (LexB) with 40 water molecules. This molecular disorder is probably attributable to a weakening of binding to the AATT site occasioned by the imidazole-for-pyrrole substitution.

Antibiotics, Antineoplastic↗
PubMed1995

Refinement of netropsin bound to DNA: bias and feedback in electron density map interpretation.

The X-ray crystal structure of the complex of the B-DNA dodecamer CGCGAATTCGCG with the antitumor drug netropsin has been reexamined to locate the drug accurately for computer-based drug design. The optimum solution is with the drug centered in the AATT region of the minor groove, making three good bifurcated hydrogen bonds with adenine N3 and thymine O2 atoms along the floor of the groove. Pyrrole rings of netropsin are packed against the C2 positions of adenines, leaving no room for the amine group of guanine and, hence, providing a structural rationale for the A.T specificity of netropsin. An alternative positioning in which the drug is shifted along the minor groove by ca. one-half base pair step is rejected on the basis of free R factor calculations and the appearance of the original drug-free difference maps. Final omit maps, although of more pleasing appearance, are not a dependable means of discriminating between right and wrong structures. The shifted alternative drug position ignores potential hydrogen bonding along the floor of the groove, provides no explanation for netropsin's observed A.T specificity, and is contradicted by NMR results [Patel, D. J. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 6424].

Base Sequence↗
PubMed1995

Crystal structure of C-T-C-T-C-G-A-G-A-G. Implications for the structure of the Holliday junction.

The structure of the B-DNA decamer of sequence C-T-C-T-C-G-A-G-A-G shows a crossed arrangement of helices in the C2 crystal lattice. This is the fourth example of a crossed arrangement of B-DNA oligomers in a crystal, and in spite of the fact that each of these four crystallizes in a different lattice, all have nearly identical structures at the crossing contact. This ubiquitous crossing arrangement may be used to generate a structure for the Holliday junction that is fully consistent with the available physical data.

Base Sequence↗
PubMed1995

Design of B-DNA cross-linking and sequence-reading molecules.

We report the design of hybrid molecules to bind in the minor groove of B-DNA, which combine DNA alkylating and cross-linking ability for increased chemotherapeutic efficacy, with sequence specificity, to minimize side effects. Optimal linkage geometries have been determined for the synthesis of bis-anthramycin and anthramycin-netropsin hybrid molecules. Earlier studies on linked drugs have typically been based on molecular mechanics calculations. This work, in contrast, uses the observed crystal structures of a netropsin/DNA complex and a new anthramycin/DNA complex to determine the exact spacing between two individual drugs when bound in the minor groove of B-DNA. Molecular linkers then are designed and tested between these two experimental positions, to form a chimeric or bis-linked compound molecule. A linked anthramycin-netropsin molecule has been designed specifically to target the polypurine tract second-strand primer site of the reverse transcriptase of HIV-1.

Base Sequence↗
PubMed1994

Bending and curvature calculations in B-DNA.

A simple program, BEND, has been written to calculate the magnitude of local bending and macroscopic curvature at each point along an arbitrary B-DNA sequence, using any desired bending model that specifies values of twist, roll and tilt as a function of sequence. The program has been used to evaluate six different DNA bending models in three categories. Two are bent non-A-tract models: (a) A new model based on the nucleosome positioning data of Satchwell et al 1986 (J. Mol. Biol. 191, 659-675), (b) The model of Calladine et al 1988 (J. Mol. Biol. 201, 127-137). Three are bent A-tract models: (c) The wedge model of Bolshoy et al 1991 (Proc. Natl. Acad. Sci. USA 88, 2312-2316), (d) The model of Cacchione et al 1989 (Biochem. 28, 8706-8713), (e) A reversed version of model (b). The last is a junction model: (f) The model of Koo & Crothers 1988 (Proc. Natl. Acad. Sci. USA 85, 1763-1767). Although they have widely different assumptions and values for twist, roll and tilt, all six models correctly predict experimental A-tract curvature as measured by gel retardation and cyclization kinetics, but only the new nucleosome positioning model is successful in predicting curvature in regions containing phased GGGCCC sequences. This model--showing local bending at mixed sequence DNA, strong bends at the sequence GGC, and straight, rigid A-tracts--is the only model consistent with both solution data from gel retardation and cyclization kinetics and structural data from x-ray crystallography.

Base Sequence↗