Iron(II) EDTA used to measure the helical twist along any DNA molecule.

A new method has been devised to measure the number of base pairs per helical turn along any DNA molecule in solution. A DNA restriction fragment is adsorbed onto crystalline calcium phosphate, fragmented by reaction with iron(II) EDTA, and subjected to electrophoresis on a denaturing polyacrylamide gel. A modulated cutting pattern results, which gives directly the helical periodicity of the DNA molecule. A 150-base pair sequence directly upstream of the thymidine kinase gene of the type 1 herpes simplex virus was found to have an overall helical twist of 10.5 base pairs per turn, which is characteristic of the B conformation of DNA. In addition, purines 3' to pyrimidines showed lower than expected reactivity toward the iron cutting reagent, which is evidence for sequence-dependent variability in DNA conformation.

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.

High-resolution footprints of the DNA-binding domain of Epstein-Barr virus nuclear antigen 1.

The DNA-binding domain of Epstein-Barr virus nuclear antigen 1 was found by hydroxyl radical footprinting to protect backbone positions on one side of its DNA-binding site. The guanines contacted in the major groove by the DNA-binding domain of Epstein-Barr virus nuclear antigen 1 were identified by methylation protection. No difference was found in the interaction of the DNA-binding domain of Epstein-Barr virus nuclear antigen 1 with tandemly repeated and overlapping binding sites.

Mapping functional regions of transcription factor TFIIIA.

Functional deletion mutants of the trans-acting factor TFIIIA, truncated at both ends of the molecule, have been expressed by in vitro transcription of a cDNA clone and subsequent cell-free translation of the synthetic mRNAs. A region of TFIIIA 19 amino acids or less, near the carboxyl terminus, is critical for maximal transcription and lies outside the DNA-binding domain. The elongated protein can be aligned over the internal control region (ICR) of the Xenopus 5S RNA gene with its carboxyl terminus oriented toward the 5' end of the gene and its amino terminus oriented toward the 3' end of the gene. The nine "zinc fingers" and the linkers that separate them comprise 80% of the protein mass and correspond to the DNA-binding domain of TFIIIA. The zinc fingers near the amino terminus of the protein contribute more to the overall binding energy of the protein to the ICR than do the zinc fingers near the carboxyl end. The most striking feature of TFIIIA is its modular structure. This is demonstrated by the fact that each zinc finger binds to just one of three short nucleotide sequences within the ICR.

A single amino acid change in CUP2 alters its mode of DNA binding.

CUP2 is a copper-dependent transcriptional activator of the yeast CUP1 metallothionein gene. In the presence of Cu+ and Ag+) ions its DNA-binding domain is thought to fold as a cysteine-coordinated Cu cluster which recognizes the palindromic CUP1 upstream activation sequence (UASc). Using mobility shift, methylation interference, and DNase I and hydroxyl radical footprinting assays, we examined the interaction of wild-type and variant CUP2 proteins produced in Escherichia coli with the UASc. Our results suggest that CUP2 has a complex Cu-coordinated DNA-binding domain containing different parts that function as DNA-binding elements recognizing distinct sequence motifs embedded within the UASc. A single-amino-acid substitution of cysteine 11 with a tyrosine results in decreased Cu binding, apparent inactivation of one of the DNA-binding elements and a dramatic change in the recognition properties of CUP2. This variant protein interacts with only one part of the wild-type site and prefers to bind to a different half-site from the wild-type protein. Although the variant has about 10% of wild-type DNA-binding activity, it appears to be completely incapable of activating transcription.

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 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.

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.

DNA footprinting with hydroxyl radical.

High resolution images of proteins bound to DNA can be achieved through the use of a simple inorganic chemical system that produces the hydroxyl radical.

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.

Structural details of an adenine tract that does not cause DNA to bend.

Runs of adenines (adenine tracts) have been implicated as the main determinant of sequence-directed DNA bending. The most widely used experimental test for bending relies on the observation that bent DNA migrates more slowly than straight DNA on a polyacrylamide electrophoresis gel. It was shown recently that the polymer (GTTTTAAAAC)n runs with normal mobility on a gel, whereas (GAAAATTTTC)n runs more slowly and thus appears to be strongly bent. The observation that these similar sequences, which differ only in the order of the adenine and thymine tracts, adopt such different shapes offers a stringent test of theories to explain DNA bending. Although the wedge model for DNA bending has recently been elaborated to explain the gel mobilities of these molecules, we wished to determine experimentally the structural basis for the difference in bending. We report here measurements of the frequency of cleavage by the hydroxyl radical at each nucleotide of cloned versions of the two polymers (see Fig. 1). We show that the TTTTAAAA sequence does not display the cleavage pattern that is associated with bent DNA, whereas the AAAATTTT sequence does. The observed sequence dependence of the cleavage pattern of an adenine tract is at odds with current models for DNA bending, which assume that adenine tracts always adopt the same conformation.

Hydroxyl radical "footprinting": high-resolution information about DNA-protein contacts and application to lambda repressor and Cro protein.

A method has been developed for making "footprints" of proteins bound to DNA. The hydroxyl radical, generated by reduction of hydrogen peroxide by iron(II), is the reagent used to cut the DNA. Hydroxyl radical breaks the backbone of DNA with almost no sequence dependence, so all backbone positions may be monitored for contact with protein. In addition to defining the DNA sequence in contact with the protein, hydroxyl radical footprints embody structural information about the DNA-protein complex. For example, hydroxyl radical footprints of the bacteriophage lambda repressor and Cro protein show directly that these proteins are bound to only one side of the DNA helix. Additional contacts of lambda repressor and Cro protein with DNA, not observed by other chemical footprinting methods, are revealed by hydroxyl radical footprinting.

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.

The unusual conformation adopted by the adenine tracts in kinetoplast DNA.

To determine the structural features responsible for the curvature of kinetoplast DNA, we studied 13 adenine tracts in Crithidia fasciculata kinetoplast DNA. The structures of the A tracts were analyzed by cutting the DNA with hydroxyl radical. Reactivity of hydroxyl radical toward the DNA backbone progressively decreased in the 5'----3' direction of each A tract. The cutting pattern of the T-rich strand was offset by 1 or 2 bp from the pattern on the A-rich strand. An A tract in a restriction fragment from plasmid pBR322 had the same cutting pattern as the kinetoplast A tracts. We interpret these experiments to show that in A tracts the width of the minor groove decreases smoothly from the 5'----3' end of the A tract.

Ethidium bromide changes the nuclease-sensitive DNA binding sites of the antitumor drug cis-diamminedichloroplatinum(II).

Exonuclease III has been shown previously to reveal the binding sites of the antitumor drug cis-diamminedichloroplatinum(II) on DNA. Pretreatment of the same DNA with the intercalator ethidium bromide causes new platinum binding sites to be detected by the exonuclease III method. In particular, a 5'd(G6-D-G2)3' sequence in a 165-base-pair restriction fragment of plasmid pBR322 becomes a preferred site for exonuclease III-detectable cis-diamminedichloroplatinum(II)binding. This switching of nuclease-sensitive platinum binding to new sites by the influence of another drug, ethidium bromide, offers an explantation at the molecular level for the phenomenon of synergism in combination chemotherapy.