The roles of specific template nucleosides in the formation of stable transcription complexes by Escherichia coli RNA polymerase.

We have examined the effects of removing individual template nucleosides on promoter escape by Escherichia coli RNA polymerase in vitro. The ability of DNA templates containing random single nucleoside gaps generated by hydroxyl radical treatment to support the production of stable ternary transcription complexes was analyzed. On two templates containing different promoter and initial transcribed regions, we found that removal of nucleosides on the template strand in the region from -13 to at least +8 relative to the transcription start site interfered with ternary complex formation. The downstream border of this region varied for the two templates, suggesting an effect of the specific nucleotide sequence on the stability of intermediates in the promoter escape process. On the nontemplate strand, removal of nucleosides in the vicinity of the -10 consensus promoter element interfered with escape, whereas removal of nucleosides in the vicinity of the transcription start site actually enhanced the yield of ternary complexes. On one template, removal of nucleosides in an A-tract containing region upstream of the promoter caused a significant decrease in promoter escape, consistent with previous suggestions that contacts between this region and the RNA polymerase play a role in promoter binding and/or initiation.

Nucleosome structural features and intrinsic properties of the TATAAACGCC repeat sequence.

Nucleosomes, the fundamental building blocks of chromatin, play an architectural role in ensuring the integrity of the genome and act as a regulator of transcription. Intrinsic properties of the underlying DNA sequence, such as flexibility and intrinsic bending, direct the formation of nucleosomes. We have earlier identified genomic nucleosome-positioning sequences with increased in vitro ability for nucleosome formation. One group of sequences bearing a 10-base pair consensus repeat sequence of TATAAACGCC had the highest reported nucleosome affinity from genomic material. Here, we report the intrinsic physical properties of this sequence and the structural details of the nucleosome it forms, as analyzed by footprinting techniques. The minor groove is buried toward the histone octamer at the AA steps and facing outwards at the CC steps. By cyclization kinetics, the overall helical repeat of the free DNA sequence was found to be 10.5 base pairs/turn. Our experiments also showed that this sequence is highly flexible, having a J-factor 25-fold higher than that of random sequence DNA. In addition, the data suggest that twist flexibility is an important determinant for translational nucleosome positioning, particularly over the dyad region.

The yeast transcription factor Mac1 binds to DNA in a modular fashion.

Mac1 is a metalloregulatory protein that regulates expression of the high affinity copper transport system in the yeast Saccharomyces cerevisiae. Under conditions of high copper concentration, Mac1 represses transcription of genes coding for copper transport proteins. Mac1 binds to DNA sequences called copper response elements (CuREs), which have the consensus sequence 5'-TTTGC(T/G)C(A/G)-3'. Mac1 contains two zinc binding sites, a copper binding site, and the sequence motif RGRP, which has been found in other proteins to mediate binding to the minor groove of A/T-rich sequences in DNA. We have used hydroxyl radical footprinting, missing nucleoside, and methylation interference experiments to investigate the structure of the complex of the DNA binding domain of Mac1 (called here Mac1(t)) with the two CuRE sites found in the yeast CTR1 promoter. We conclude from these experiments that Mac1(t) binds in a modular fashion to DNA, with its RGRP AT-hook motif interacting with the TTT sequence at the 5' end of the CTR1 CuRE site, and with another DNA-binding module(s) binding in the adjacent major groove in the GCTCA sequence.

DNA strand breaking by the hydroxyl radical is governed by the accessible surface areas of the hydrogen atoms of the DNA backbone.

Despite extensive study, there is little experimental information available as to which of the deoxyribose hydrogen atoms of duplex DNA reacts most with the hydroxyl radical. To investigate this question, we prepared a set of double-stranded DNA molecules in which deuterium had been incorporated specifically at each position in the deoxyribose of one of the four nucleotides. We then measured deuterium kinetic isotope effects on the rate of cleavage of DNA by the hydroxyl radical. These experiments demonstrate that the hydroxyl radical reacts with the various hydrogen atoms of the deoxyribose in the order 5' H > 4' H > 3' H approximately 2' H approximately 1' H. This order of reactivity parallels the exposure to solvent of the deoxyribose hydrogens. Our work therefore reveals the structural basis of the reaction of the hydroxyl radical with DNA. These results also provide information on the mechanism of DNA damage caused by ionizing radiation as well as atomic-level detail for the interpretation of hydroxyl radical footprints of DNA-protein complexes and chemical probe experiments on the structure of RNA and DNA in solution.

The DNA binding specificity of engrailed homeodomain.

The engrailed gene of Drosophila melanogaster is an integral member of the highly complex cascade which results in a fully developed fruitfly. The gene product of engrailed contains a homeodomain which is responsible for DNA binding via a helix-turn-helix motif. The crystal structure of this 60 amino acid residue domain complexed to DNA is analogous to structures of other homeodomain-DNA complexes, consistent with the high degree of sequence conservation within both protein and DNA. Despite the high degree of homology, homeodomains do exhibit distinct preferences for certain DNA sequences. Such specificity may be at least partly responsible for the interactions necessary for normal development. Using the hydroxyl radical as a chemical probe, we have examined complexes of Engrailed homeodomain with several DNA sequences to determine the protein's binding specificity in solution. We find that Engrailed forms a single, specific complex with a unique DNA binding site which is analogous to the complex seen in the co-crystal structure. In contrast, our chemical probe experiments show that the binding site of Engrailed that was determined by in vitro selection and that also was present in the co-crystal structure contains two possible binding sites. Modification of the sequence of this site to yield single binding sites removes the ambiguity, and results in two different, well-behaved Engrailed-DNA complexes. Our results underscore the utility of chemical probe experiments for defining the variety of modes of interaction of proteins with DNA that can occur in solution, but that might not be apparent in a crystal structure.

The solution structure of the amino-terminal HHCC domain of HIV-2 integrase: a three-helix bundle stabilized by zinc.

BACKGROUND: Integrase mediates a crucial step in the life cycle of the human immunodeficiency virus (HIV). The enzyme cleaves the viral DNA ends in a sequence-dependent manner and couples the newly generated hydroxyl groups to phosphates in the target DNA. Three domains have been identified in HIV integrase: an amino-terminal domain, a central catalytic core and a carboxy-terminal DNA-binding domain. The amino-terminal region is the only domain with unknown structure thus far. This domain, which is known to bind zinc, contains a HHCC motif that is conserved in retroviral integrases. Although the exact function of this domain is unknown, it is required for cleavage and integration. RESULTS: The three-dimensional structure of the amino-terminal domain of HIV-2 integrase has been determined using two-dimensional and three-dimensional nuclear magnetic resonance data. We obtained 20 final structures, calculated using 693 nuclear Overhauser effects, which display a backbone root-mean square deviation versus the average of 0.25 A for the well defined region. The structure consists of three alpha helices and a helical turn. The zinc is coordinated with His 12 via the N epsilon 2 atom, with His16 via the N delta 1 atom and with the sulfur atoms of Cys40 and Cys43. The alpha helices form a three-helix bundle that is stabilized by this zinc-binding unit. The helical arrangement is similar to that found in the DNA-binding domains of the trp repressor, the prd paired domain and Tc3A transposase. CONCLUSION: The amino-terminal domain of HIV-2 integrase has a remarkable hybrid structure combining features of a three-helix bundle fold with a zinc-binding HHCC motif. This structure shows no similarity with any of the known zinc-finger structures. The strictly conserved residues of the HHCC motif of retroviral integrases are involved in metal coordination, whereas many other well conserved hydrophobic residues are part of the protein core.

Quantitative analysis of electrophoresis data: novel curve fitting methodology and its application to the determination of a protein-DNA binding constant.

A computer program, GelExplorer, which uses a new methodology for obtaining quantitative information about electrophoresis has been developed. It provides a straightforward, easy-to-use graphical interface, and includes a number of features which offer significant advantages over existing methods for quantitative gel analysis. The method uses curve fitting with a nonlinear least-squares optimization to deconvolute overlapping bands. Unlike most curve fitting approaches, the data is treated in two dimensions, fitting all the data across the entire width of the lane. This allows for accurate determination of the intensities of individual, overlapping bands, and in particular allows imperfectly shaped bands to be accurately modeled. Experiments described in this paper demonstrate empirically that the Lorentzian lineshape reproduces the contours of an individual gel band and provides a better model than the Gaussian function for curve fitting of electrophoresis bands. Results from several fitting applications are presented and a discussion of the sources and magnitudes of uncertainties in the results is included. Finally, the method is applied to the quantitative analysis of a hydroxyl radical footprint titration experiment to obtain the free energy of binding of the lambda repressor protein to the OR1 operator DNA sequence.

Effect of the crystallizing agent 2-methyl-2,4-pentanediol on the structure of adenine tract DNA in solution.

The hydroxyl radical cleavage pattern of bent DNA containing phased adenine tracts has a sinusoidal pattern. Changes in the cleavage pattern of the sequence A5N5 in the presence of 2-methyl-2,4-pentanediol (MPD) demonstrate that MPD has an effect on the structure of a bent A-tract region of DNA. The effect of MPD on DNA structure appears to be limited to bent A-tract regions, as we find no significant changes in the cleavage pattern of flanking regions, mixed-sequence DNA, or the unbent A-tract sequence T4A4N2.

Chemical probe and missing nucleoside analysis of Flp recombinase bound to the recombination target sequence.

The Flp protein catalyzes a site-specific recombination reaction between two 47 bp DNA sites without the assistance of any other protein or cofactor. The Flp recognition target (FRT) site consists of three nearly identical sequences, two of which are separated by an 8 bp spacer sequence. In order to gain insight into this remarkable protein-DNA interaction we used a variety of chemical probe methods and the missing nucleoside experiment to examine Flp binding. Hydroxyl radical footprints of Flp bound to a recombinationally-competent site fall on opposite faces of canonical B-DNA. The 8 bp spacer region between the two Flp binding sites becomes reactive towards 5-phenyl-1,10-phenanthroline.copper upon Flp binding, indicating that once bound by Flp, this segment of DNA is not in the B-form. Missing nucleoside analysis reveals that within each binding site the presence of two nucleosides on the top strand and four on the bottom, are required for formation of a fully-occupied FRT site. In contrast, loss of any nucleoside in the three binding sites in the FRT interferes with formation of lower-occupancy complexes. DNA molecules with gaps in the 8 bp spacer region are over-represented in complexes with either two or three binding sites occupied by Flp, evidence that DNA flexibility facilitates the cooperative interaction of Flp protomers bound to a recombinationally-active site.

Homeodomain proteins: what governs their ability to recognize specific DNA sequences?

Deformed (Dfd) and Ultrabithorax (Ubx) are homeodomain proteins from Drosophila melanogaster that exert regulatory effects on gene expression by binding to specific target sites in the fly genome using a helix-turn-helix (HTH) motif. The recognition helices of these two proteins are almost identical and the DNA sequences they recognize are similar, containing a conserved TAAT core sequence flanked by a somewhat variable sequence. Yet the in vivo functions of the two proteins are quite different. We have used the homeodomains of these two proteins and in vitro selected DNA binding sites to characterize the structural details of homeodomain binding to DNA and to understand the basis for the differences in sequence specificity between homeodomains with similar recognition helices. We have employed hydroxyl radical cleavage of DNA to study the positioning of the proteins on the binding sites and have analyzed the effects of missing nucleosides and purine methylation on homeodomain binding. Our results indicate that the positioning of the Ubx and Dfd homeodomains on their binding sites is consistent with reported structures of other homeodomain/DNA complexes. Dfd and Ubx bind to DNA with the recognition helix in the major groove 3' to the TAAT core sequence and the N-terminal arm in the adjacent minor groove. However, we observe striking differences between the two homeodomains in their specific interactions with DNA. Missing nucleosides within the selected binding sites have differential effects on protein binding, which are dependent on the identity of the homeodomain. Differences at the 3' end of the binding site on the top strand indicate that the N-terminal arm of a homeodomain is capable of distinguishing an A.T base-pair from T.A in the minor groove. Specific orientation of the N-terminal arm within the binding site appears to vary between the homeodomains and influences the interaction of the recognition helix with the major groove.

Cleavage by calicheamicin gamma 1I of DNA in a nucleosome formed on the 5S RNA gene of Xenopus borealis.

The cleavage by calicheamicin gamma 1I (CLM gamma 1I) of a nucleosome formed on the 5S RNA gene of Xenopus borealis was studied in vitro as a first step toward the understanding of CLM gamma 1I-chromatin interactions within the cell. The drug does not cleave in the region of the dyad axis of the nucleosome. Outside of this region, double-stranded cleavage occurs with a periodicity of 10-11 bp. The sites of cleavage correspond to DNA sequences facing outward in the nucleosome. The drug shows some sequence preference of cleavage within these accessible sites. The predominant cleavage event within this nucleosome occurs at -1 helical turn from the dyad axis. This site constitutes a "hot spot" for CLM gamma 1I cleavage within the 5S nucleosome. We observe an overall footprinting effect whereby the drug preferentially cleaves DNA located outside the nucleosome core (analogous to the linker DNA of chromatin) as compared to cleavage within the nucleosome core. We discuss the importance of accessibility, structural deformations of DNA within the nucleosome, and steric constraints posed by sequence, in the recognition and cleavage of nucleosomal DNA by calicheamicin.

Hydroxyl radical footprinting of calicheamicin. Relationship of DNA binding to cleavage.

The binding to DNA by calicheamicin epsilon (CLM epsilon), the rearranged and reduced product of the diynene antitumor antibiotic calicheamicin gamma 1I (CLM gamma 1I), was studied using the method of hydroxyl radical footprinting. The drug binding sites determined in this way were compared to locations of double-stranded DNA cleavage by thiol-activated CLM gamma 1I. The results of these experiments show that CLM epsilon lies in the minor groove in an extended conformation protecting approximately four nucleotides on each strand of DNA. Sites of CLM epsilon binding correlate to sites of CLM gamma 1I cleavage with protection by CLM epsilon occurring mainly to the 3' side of the site of C5' hydrogen abstraction. From these results, it is possible to propose global structures of the drug/DNA complexes such that the oligosaccharide side chain is arrayed to the 3' side of the site of C5' hydrogen abstraction. This conclusion is entirely consistent with the results of recent atom-transfer experiments [Hangeland, J.J., De Voss, J.J., Heath, J.A., Townsend, C.A., Ding, W.-D., Ashcroft, J., & Ellestad, G.A. (1992) J. Am. Chem. Soc. 114, 9200-9202]. Somewhat greater protection on the strand undergoing C5' hydrogen abstraction was observed to the 5' side of the site of attack owing presumably to proximity of the methyl carbamate portion of the drug with DNA. Overall, binding is seen where cleavage is seen in accord with thermodynamics of drug association to DNA being important in determining the sites of cleavage.

Sequence-specific cleavage of DNA via nucleophilic attack of hydrogen peroxide, assisted by Flp recombinase.

Hydrogen peroxide is capable of effecting the cleavage of a specific phosphodiester bond in DNA, when used in concert with the recombinase enzyme Flp from Saccharomyces cerevisiae. This cleavage is not caused by oxidative damage of the DNA backbone but instead is the result of nucleophilic attack by peroxide. A single phosphorus-oxygen bond is broken in the reaction. Cleavage of DNA by peroxide also occurs with an inactive mutant of Flp in which the active site nucleophile tyrosine has been replaced by phenylalanine. Besides providing information on the mechanism of strand cleavage by Flp, these results may contribute to the development of new synthetic DNA cleavage reagents that act by hydrolytic and not radical chemistry.

Structure of DNA in a nucleosome core at high salt concentration and at high temperature.

We have used hydroxyl radical cleavage of DNA to probe the organization of the nucleosome core at high salt concentration and high temperature. The rotational and translational positioning of a DNA fragment, containing part of the Xenopus borealis 5S RNA gene, on the histone octamer is maintained between salt concentrations of 0.0 and 0.8 M NaCl and between temperatures of 0 and 75 degrees C. These results provide evidence that the energy of bending DNA around the nucleosome is independent of salt concentration and temperature in this range. They illustrate the severe energetic requirements necessary to displace DNA from previously organized contacts with histones in the nucleosome core.

How the structure of an adenine tract depends on sequence context: a new model for the structure of TnAn DNA sequences.

We present a new model to explain the bending and local structural properties of TnAn sequences in DNA. Current models suggest that an adenine tract has the same unusual structure when found in a TnAn sequence as it has when surrounded by mixed-sequence B-DNA. On the basis of hydroxyl radical cleavage patterns of several TnAn sequences, we instead propose that the T2A2 or T3A3 core of such sequences is B-DNA-like but that adenines and thymines outside of this core, if sufficient in number, can form the unusual structure adopted by adenine tracts surrounded by mixed sequence DNA. We pursued further the structure of T7A7N7, a molecule which exhibits reduced electrophoretic mobility on native polyacrylamide gels and is therefore presumed to be bent. We attempted to mimic the structure of T7A7N7 that was predicted by our model by designing two new sequences, one in which the T3A3 core of T7A7N7 is substituted by six nucleotides of mixed sequence (N6) and the other in which the T2A2 core is replaced by N4. Hydroxyl radical cleavage patterns of all three molecules are nearly indistinguishable. All three molecules run anomalously slowly on a native polyacrylamide gel, with the mobility of T4N6A4N7 > T7A7N7 approximately T5N4A5N7. Analysis of the hydroxyl radical cutting pattern of T7A7N7 by Fourier transformation reveals the occurrence of an unusual structure at intervals of approximately 10 bp, a periodicity which is not evident in the sequence of the DNA.

Structure of the TFIIIA-5 S DNA complex.

The missing-nucleoside experiment, a recently developed approach for determining the positions along a DNA molecule that make energetically important contacts with protein, has been used to investigate the structure of the complex of transcription factor IIIA with a somatic 5 S RNA gene from Xenopus borealis. We detect three distinct regions of the 5 S promoter that are contacted by TFIIIA, corresponding to the A-box, intermediate element and C-box regions previously identified by mutagenesis experiments. The advantage of the missing-nucleoside experiment over mutagenesis is that additional information, directly related to the structure of the complex, is obtained. Of most importance is that contacts to each strand of DNA are determined independently, and can be assigned unambiguously as interactions with TFIIIA. Throughout the binding site the strongest contacts are made with the non-coding strand of the 5 S gene. The two groups of contacts at either end of the binding site (boxes A and C) are comprised of sets of approximately ten contiguous nucleosides for which the contacts are reflected, without stagger, from one strand to the other. In contrast, contacts in the center of the promoter (the intermediate element) are staggered about five base-pairs in the 5' direction with respect to each strand. These results, when analyzed in conjunction with the hydroxyl-radical footprint of the complex, support a model in which TFIIIA wraps around the DNA in the major groove of the helix for one turn at the two ends of the complex in boxes A and C, and lies on one side of the DNA helix in the center of the complex at the intermediate element.

The histone core exerts a dominant constraint on the structure of DNA in a nucleosome.

We have examined the structures of unique sequence, A/T-rich DNAs that are predicted to be relatively rigid [oligo(dA).oligo(dT)], flexible [oligo[d(A-T)]], and curved, using the hydroxyl radical as a cleavage reagent. A 50-base-pair segment containing each of these distinct DNA sequences was placed adjacent to the T7 RNA polymerase promoter, a sequence that will strongly position nucleosomes. The final length of the DNA fragments was 142 bp, enough DNA to assemble a single nucleosome. Cleavage of DNA in solution, while bound to a calcium phosphate crystal, and after incorporation into a nucleosome is examined. We find that the distinct A/T-rich DNAs have very different structural features in solution and helical periodicities when bound to a calcium phosphate. In contrast, the organization of the different DNA sequences when associated with a histone octamer is very similar. We conclude that the histone core exerts a dominant constraint on the structure of DNA in a nucleosome and that inclusion of these various unique sequences has only a very small effect on overall nucleosome stability and structure.

DNA footprinting with the hydroxyl radical.

The Fenton reaction of iron(II) EDTA with hydrogen peroxide, performed in the presence of ascorbate ion, has proven to be useful as a probe of structure in DNA systems. Two aspects of this chemistry are discussed: the identity of the active DNA cleaving agent produced by this reagent, and the application of the Fenton reaction to the determination of the structure of the Holliday junction, the four-stranded DNA molecule that is a key intermediate in recombination. The cleavage pattern of the Holliday junction has pseudo-twofold symmetry, putting important constraints on possible structures.