Replacement of an NH(3) by an iminoether in transplatin makes an antitumor drug from an inactive compound.

To investigate the modifications of antitumor activity and DNA binding mode of transplatin after replacement of one nonleaving group NH(3) by an iminoether group, trans-[PtCl(2)(Z-HN=C(OMe)Me)(NH(3)] and trans-[PtCl(2)(E-HN=C(OMe)Me)(NH(3)] complexes (differing in the Z or E configuration of iminoether, and abbreviated mixed Z and mixed E, respectively), have been synthesized. In a panel of human tumor cell lines, both mixed Z and mixed E show a cytotoxic potency higher than that of transplatin, the mean IC(50) values being 103, 37, and 215 microM, respectively. In vivo mixed Z is more active and less toxic than mixed E in murine P388 leukemia and retains its efficacy against SK-OV-3 human cancer cell xenograft in nude mice. In the reaction with naked DNA, mixed Z forms monofunctional adducts that do not evolve into intrastrand cross-links but close slowly into interstrand cross-links between complementary guanine and cytosine residues. The monofunctional mixed Z adducts are removed by thiourea and glutathione. The interstrand cross-links behave as hinge joints, increasing the flexibility of DNA double helix. The mixed Z, transplatin, and cisplatin interstrand cross-links, as well as mixed Z monofunctional adducts are not specifically recognized by HMG1 protein, which was confirmed to be able to specifically recognize cisplatin d(GpG) intrastrand cross-links. These data demonstrate that the DNA interaction properties of the antitumor-active mixed Z are very similar to those of transplatin, thus suggesting that clinical inactivity of transplatin could not depend upon its peculiar DNA binding mode.

Transplatin-modified oligonucleotides as modulators of gene expression.

Transplatin [trans-diamminedichloroplatinum(II)], contrary to its stereoisomer cisplatin, is clinically inactive. However, like cisplatin, it binds to DNA. In the first part of this review, some results on the interactions between transplatin and double-stranded DNA are presented. The major bifunctional adducts are interstrand cross-links. Intrastrand cross-links are not formed. On the other hand, intrastrand cross-links are formed in the reaction between single-stranded DNA and transplatin. Some properties of the intrastrand cross-links at GNG sites (N is a nucleotide) are described. The (G1,G3)-intrastrand cross-links rearrange into interstrand cross-links as soon as the platinated oligonucleotides are paired with their complementary strands. The linkage isomerization reaction is exclusively triggered by the formation of a double helix. The potential use of these platinated oligonucleotides to block the cellular machinery specifically and irreversibly is discussed.

HMG boxes of DSP1 protein interact with the rel homology domain of transcription factors.

Formation of the dorsoventral axis in Drosophila melanogaster is mediated through control of the expression of several genes by the morphogen Dorsal. In the ventral part of the embryo Dorsal activates twist and represses zen amongst others. Recently, several proteins have been shown to assist Dorsal in the repression of zen, one of which is DSP1, a HMG box protein that was isolated as a putative co-repressor of Dorsal. In this report we used a DSP1 null mutant to ascertain in vivo the involvement of DSP1 in Dorsal-mediated repression of zen but not in the activation of twist. We show that Dorsal has the ability to interact with DSP1 in vitro as well as with rat HMG1. Using truncated versions of the proteins we located the domains of interaction as being the HMG boxes for DSP1 and HMG1 and the Rel domain for Dorsal. Finally, studies of the zen DNA binding properties of Dorsal and another related Rel protein (Gambif1 from Anopheles gambiae) revealed that their DNA binding affinities were increased in the presence of DSP1 and HMG1.

Interstrand cross-links of cisplatin induce striking distortions in DNA.

In the reaction between cellular DNA and cisplatin, different bifunctional adducts are formed including intrastrand and interstrand cross-links. The respective role of these lesions in the cytotoxicity of the drug is not yet elucidated. This paper deals with the current knowledge on cisplatin interstrand cross-links and presents results on the formation, stability and structure of these adducts. A key step in the studies of these lesions is the recent determination of solution and crystallographic structures of double-stranded oligonucleotides containing a unique interstrand cross-link. The DNA distortions induced by this adduct exhibit unprecedented features such as the location of the platinum residue in the minor groove, the extrusion of the cytosines of the cross-linked d(GpC).d(GpC) site, the bending of the helix axis towards the minor groove and a large DNA unwinding. In addition to a detailed determination of the distortions, the high resolution of the crystal structure allowed us to locate the water molecules surrounding the adduct. The possible implications of this structure for the chemical properties and the cellular processing of cisplatin interstrand cross-links are discussed.

Structural recognition and distortion by the DNA junction-resolving enzyme RusA.

RusA is a relatively small DNA junction-resolving enzyme of lambdoid phage-origin. Many of the physical characteristics of this enzyme are similar to those of junction-resolving enzymes of different origins. RusA binds to DNA junctions as a dimer, with a dissociation constant of 2 to 7 nM. RusA also exists in dimeric form in free solution, with a half time for subunit exchange of 4.2 minutes. We find that RusA can cleave both fixed junctions and those that can undergo a number of steps of branch migration, and confirm that the enzyme exhibits a strong preference for cleavage 5' to a CpC sequence. We have isolated a mutant protein, RusA D70N, that is completely inactive in cleavage while binding normally to DNA junctions, suggesting a role for aspartate 70 in the cleavage reaction. Constraining the conformation of the junction by means of tethering the helical ends leads to a marked reduction in cleavage rate by RusA, suggesting that the structure must be altered for cleavage. Using comparative gel electrophoresis we find that the global structure of the DNA junction is altered on RusA binding, into a structure that is different from any that is formed by the free junction. Moreover, the structure of the complex is the same irrespective of the presence or absence of magnesium ions. Thus, like all the junction-resolving enzymes, RusA both recognises and distorts the structure of DNA junctions.

Recognition and manipulation of branched DNA structure by junction-resolving enzymes.

The junction-resolving enzymes are a class of nucleases that introduce paired cleavages into four-way DNA junctions. They are important in DNA recombination and repair, and are found throughout nature, from eubacteria and their bacteriophages through to higher eukaryotes and their viruses. These enzymes exhibit structure-selective binding to DNA junctions; although cleavage may be more or less sequence-dependent, binding affinity is purely related to the branched structure of the DNA. Binding and cleavage events can be separated for a number of the enzymes by mutagenesis, and mutant proteins that are defective in cleavage while retaining normal junction-selective binding have been isolated. Critical acidic residues have been identified in several resolving enzymes, suggesting a role in the coordination of metal ions that probably deliver the hydrolytic water molecule. The resolving enzymes all bind to junctions in dimeric form, and the subunits introduce independent cleavages within the lifetime of the enzyme-junction complex to ensure resolution of the four-way junction. In addition to recognising the structure of the junction, recent data from four different junction-resolving enzymes indicate that they also manipulate the global structure. In some cases this results in severe distortion of the folded structure of the junction. Understanding the recognition and manipulation of DNA structure by these enzymes is a fascinating challenge in molecular recognition.

Near-simultaneous DNA cleavage by the subunits of the junction-resolving enzyme T4 endonuclease VII.

In common with a number of other DNA junction-resolving enzymes, endonuclease VII of bacteriophage T4 binds to a four-way DNA junction as a dimer, and cleaves two strands of the junction. We have used a supercoil-stabilized cruciform substrate to probe the simultaneity of cleavage at the two sites. Active endonuclease VII converts the supercoiled circular DNA directly into linear product, indicating that the two cleavage reactions must occur within the lifetime of the protein-junction complex. By contrast, a heterodimer of active enzyme and an inactive mutant endonuclease VII leads to the formation of nicked circular product, showing that the subunits operate fully independently.

Nucleic acid structure and recognition.

We review the global structures adopted by branched nucleic acids, including three- and four-way helical junctions in DNA and RNA. We find that some general folding principles emerge. First, all the structures exhibit a tendency to undergo pairwise coaxial helical stacking when permitted by the local stereochemistry of strand exchange. Second, metal ions generally play an important role in facilitating folding of branched nucleic acids. These principles can be applied to functionally important branched nucleic acids, such as the Holliday DNA junction of genetic recombination, and the hammerhead ribozyme in RNA.

T4 endonuclease VII. Importance of a histidine-aspartate cluster within the zinc-binding domain.

The DNA junction-resolving enzyme endonuclease VII of bacteriophage T4 contains a zinc-binding region toward the N-terminal end of the primary sequence. In the center of this 39-amino acid section (between residues 38 and 44) lies the sequence HLDHDHE, termed the His-acid cluster. Closely related sequences are found in three other proteins that have similar zinc-binding motifs. We have analyzed the function of these residues by a site-directed mutagenesis approach, modifying single amino acids and studying the properties of the resulting N-terminal protein A fusions. No sequence changes within the His-acid cluster led to a change in zinc content of the protein, indicating that these residues are not involved in the coordination of zinc. We found that the N-terminal aspartate residue (Asp-40) and the two histidine residues (His-41 and His-43) within the cluster are essential for junction-cleavage activity of the proteins. However, all sequence variations within this region generate proteins that retain their ability to bind to four-way DNA junctions (with minor changes in binding affinity in some cases) and to distort their global structure in the same manner as active enzymes. We conclude that the process of cleavage can be uncoupled from those of binding to and distortion of the junction. It is probable that some amino acid side chains of the His-acid cluster participate in the phosphodiester cleavage mechanism of endonuclease VII. The essential aspartate residue might be required for coordination of catalytic metal ions.

T4 endonuclease VII selects and alters the structure of the four-way DNA junction; binding of a resolution-defective mutant enzyme.

Bacteriophage T4 endonuclease VII is a nuclease that is selective for four-way DNA junctions and related branched DNA species. Using site-directed mutagenesis we have isolated a mutant protein (E86A) that is inactive in the cleavage of DNA junctions while retaining full selectivity of binding. Using endonuclease VII E86A we have shown: (1) The protein binds as a dimer to DNA junctions, with rapid exchange of subunits in free solution. (2) Binding to junctions is highly selective for the structure of DNA junctions; the complex is not displaced by a 1000-fold excess of duplex competitor DNA. (3) On binding endonuclease VII E86A to junctions, the configuration of the helical arms is significantly altered to a structure that is independent of the presence or absence of metal ions. We suggest a model for the structure of the junction in the protein complex. (4) The protein can bind to the junction in two stereochemically equivalent ways, depending upon the sequence of the junction. T4 endonuclease VII is a junction-selective enzyme that both recognises and manipulates the structure of its substrate.

The modular character of a DNA junction-resolving enzyme: a zinc-binding motif in bacteriophage T4 endonuclease VII.

Bacteriophage T4 endonuclease VII is one of a class of structure-selective enzymes that resolve helical branchpoints in DNA molecules. The sequence of this protein suggests a modular organisation. We have expressed a synthetic gene encoding endonuclease VII, which has been used in a directed mutagenesis exercise, with the aim of understanding the role of different sections of the protein sequence. Towards the N-terminal end of the protein lies a section of polypeptide in which four cysteine residues distributed in a CxxC--CxxC pattern co-ordinate one atom of zinc. The N-terminal section composed of amino acid residues 1 to 65 isolated from the remaining C-terminal section also binds one mole of zinc, suggesting that this region folds autonomously. Mutation shows that the outer cysteine residues are essential for zinc binding, while the inner cysteine residues are partially degenerate in that either one of the two (but not both) can be replaced while retaining some zinc. The activity as a junction-resolving enzyme correlated qualitatively with the presence of the zinc. In the C-terminal part of the protein lies a section that is 48% identical with a sequence found in the DNA repair protein T4 endonuclease V. We can replace the section of T4 endonuclease VII with the corresponding sequence from T4 endonuclease V with no change in the pattern of cleavage on four-way junctions. The evidence supports a modular construction for T4 endonuclease VII.

Structure of the four-way DNA junction and its interaction with proteins.

The four-way DNA junction is an important intermediate in recombination processes; it is, the substrate for different enzyme activities. In solution, the junction adopts a right-handed, antiparallel-stacked X-structure formed by the pairwise coaxial-stacking of helical arms. The stereochemistry is determined by the juxtaposition of grooves and backbones, which is optimal when the smaller included angle is 60 degrees. The antiparallel structure has two distinct sides with major and minor groove-characteristics, respectively. The folding process requires the binding of metal cations, in the absence of which, the junction remains extended without helix-helix stacking. The geometry of the junction can be perturbed by the presence of certain base-base mispairs or phosphodiester discontinuities located at the point of strand exchange. The four-way DNA junction is selectively cleaved by a number of resolving enzymes. In a number of cases, these appear to recognize the minor groove face of the junction and are functionally divisible into activities that recognize and bind the junction, and a catalytic activity. Some possible mechanisms for the recognition of branched DNA structure are discussed.

Fluorescence study on the non-specific binding of cyclic-AMP receptor protein to DNA: effect of pH.

The binding of the cyclic-AMP receptor protein (CRP) of Escherichia coli to a non-specific DNA fragment of 46 base pairs has been studied using fluorescence spectroscopy. The equilibrium binding constant was found to be several orders of magnitude lower than in the specific binding to a DNA fragment of the same size. The salt dependence of the equilibrium binding constant indicates that the CRP makes an identical number (8) of ion pairs to this non-specific DNA fragment in the presence and absence of cAMP. This number is larger than that previously found in the specific binding process. The effect of pH on the non-specific binding was investigated. The number of ion pairs does not vary between pH 6 and 8. From the variation of the binding constant with pH it was deduced that two histidines are involved in the binding in the absence of cAMP. These are most probably the histidines 199 of each subunit. In the presence of cAMP, only one histidine participates in the binding process, indicating an asymmetric interaction between the two subunits of the CRP and the DNA.

Specific binding of cyclic-AMP receptor protein to DNA. Effect of the sequence and of the introduction of a nick in the binding site.

The binding of Escherichia coli Cyclic AMP Receptor Protein (CRP) to several DNA fragments of about 45 base pairs, bearing the natural lactose or galactose sites, as well as several synthetic related sites, was investigated using fluorescence spectroscopy and gel retardation experiments. The salt dependence of the equilibrium binding constant indicates that CRP makes an identical number of ion pairs with the lac, lacL8 and gal sites although the binding constants are drastically different. However increasing the symmetry of the gal site leads to an increase of the number of ion pairs between the protein and the DNA. A single strand nick was introduced at the centre of a symmetrized gal site and this reduces the binding energy of CRP by about 0.6 Kcal. These results are discussed with respect to the bending constraints imposed on the DNA by the binding of CRP. The results are in agreement with the recently published crystal structure of the CRP complexed with DNA [Schutz, S.C., Shields, G.C. and Steitz, T.A., Science 253, 1001-1007 (1991)] showing that the 90 degrees bending of the DNA in the complex results from two kinks.