Lanthanide Spectroscopic Studies of the Dinuclear and Mg(II)-Dependent PvuII Restriction Endonuclease

Type II restriction enzymes are homodimeric systems that bind four to eight base pair palindromic recognition sequences of DNA and catalyze metal ion-dependent phosphodiester cleavage. While Mg(II) is required for cleavage in these enzymes, in some systems Ca(II) promotes avid substrate binding and sequence discrimination. These properties make them useful model systems for understanding the roles of alkaline earth metal ions in nucleic acid processing. We have previously shown that two Ca(II) ions stimulate DNA binding by PvuII endonuclease and that the trivalent lanthanide ions Tb(III) and Eu(III) support subnanomolar DNA binding in this system. Here we capitalize on this behavior, employing a unique combination of luminescence spectroscopy and DNA binding assays to characterize Ln(III) binding behavior by this enzyme. Upon excitation of tyrosine residues, the emissions of both Tb(III) and Eu(III) are enhanced severalfold. This enhancement is reduced by the addition of a large excess of Ca(II), indicating that these ions bind in the active site. Poor enhancements and affinities in the presence of the active site variant E68A indicate that Glu68 is an important Ln(III) ligand, similar to that observed with Ca(II), Mg(II), and Mn(II). At low micromolar Eu(III) concentrations in the presence of enzyme (10−20 μM), Eu(III) excitation 7F0 → 5D0 spectra yield one dominant peak at 579.2 nm. A second, smaller peak at 579.4 nm is apparent at high Eu(III) concentrations (150 μM). Titration data for both Tb(III) and Eu(III) fit well to a two-site model featuring a strong site (Kd = 1−3 μM) and a much weaker site (Kd ≈ 100−200 μM). Experiments with the E68A variant indicate that the Glu68 side chain is not required for the binding of this second Ln(III) equivalent; however, the dramatic increase in DNA binding affinity around 100 μM Ln(III) for the wild-type enzyme and metal-enhanced substrate affinity for E68A are consistent with functional relevance for this weaker site. This discrimination of sites should make it possible to use lanthanide substitution and lanthanide spectroscopy to probe individual metal ion binding sites, thus adding an important tool to the study of restriction enzyme structure and function

Structural Studies of Λ- and Δ-[Ru(phen)_2dppz]^(2+)Bound to d(GTCGAC)_2: Characterization of Enantioselective Intercalation

^1H and ^(31)P NMR have been applied in characterizing the enantioselective interactions between Λ- and Δ-[Ru(phen)_2dppz]^(2+) and the hexamer oligonucleotide d(GTCGAC)_2. Both isomers intercalate into the helix, and intermolecular NOEs place the Δ-isomer in the major groove. The NMR results indicate that Ru(phen)_2dppz^(2+) isomers bind to the DNA helix with a population of intercalative geometries and therefore extend earlier structural models based upon luminescence studies

Structural Studies of Λ- and Δ-[Ru(phen)_2dppz]^(2+)Bound to d(GTCGAC)_2: Characterization of Enantioselective Intercalation

^1H and ^(31)P NMR have been applied in characterizing the enantioselective interactions between Λ- and Δ-[Ru(phen)_2dppz]^(2+) and the hexamer oligonucleotide d(GTCGAC)_2. Both isomers intercalate into the helix, and intermolecular NOEs place the Δ-isomer in the major groove. The NMR results indicate that Ru(phen)_2dppz^(2+) isomers bind to the DNA helix with a population of intercalative geometries and therefore extend earlier structural models based upon luminescence studies

Enantiospecific palindromic recognition of 5'-d(CTCTAGAG)-3' by a novel rhodium intercalator: analogies to a DNA-binding protein

We are exploring principles of protein-DNA recognition through the design of model 9,10-phenanthrenequinonediimine (phi) complexes of rhodium(II1) which bind site-selectively to DNA. [Rh(phi)]^(3+) complexes intercalate in the DNA major groove through the phi ligand and upon photoactivation promote strand scission via abstraction of the deoxyribose C3‘-H atom. Here we report on the metal complex [Rh(DPB)_2phi]^(3+) (DPB = 4,4’-diphenyl-2,2’-bipyridyl) (Figure 1 ), which mimics DNA-binding proteins in DNA site-specificity and affinity

Crystal structures of Λ-[Ru(phen)2dppz]2+ with oligonucleotides containing TA/TA and AT/AT steps show two intercalation modes

The ruthenium complex [Ru(phen)2(dppz)] (where phen is a phenanthroline and dppz a dipyridyl–phenazine ligand) is known as a ‘light switch’ complex because its luminescence in solution is significantly enhanced in the presence of DNA. This property is poised to serve in diagnostic and therapeutic applications, but its binding mode with DNA needs to be elucidated further. Here, we describe the crystal structures of the L enantiomer bound to two oligonucleotide duplexes. The dppz ligand intercalates symmetrically and perpendicularly from the minor groove of the d(CCGGTACCGG)2 duplex at the central TA/TA step, but not at the central AT/AT step of d(CCGGATCCGG)2. In both structures, however, a second ruthenium complex links the duplexes through the combination of a shallower angled intercalation into the C1C2/G9G10 step at the end of the duplex, and semi-intercalation into the G3G4 step of an adjacent duplex. The TA/TA specificity of the perpendicular intercalation arises from the packing of phenanthroline ligands against the adenosine residue

Dissimilar Roles of the Four Conserved Acidic Residues in the Thermal Stability of Poly(A)-Specific Ribonuclease

Divalent metal ions are essential for the efficient catalysis and structural stability of many nucleotidyl-transfer enzymes. Poly(A)-specific ribonuclease (PARN) belongs to the DEDD superfamily of 3′-exonucleases, and the active site of PARN contains four conserved acidic amino acid residues that coordinate two Mg2+ ions. In this research, we studied the roles of these four acidic residues in PARN thermal stability by mutational analysis. It was found that Mg2+ significantly decreased the rate but increased the aggregate size of the 54 kDa wild-type PARN in a concentration-dependent manner. All of the four mutants decreased PARN thermal aggregation, while the aggregation kinetics of the mutants exhibited dissimilar Mg2+-dependent behavior. A comparison of the kinetic parameters indicated that Asp28 was the most crucial one to the binding of the two Mg2+ ions, while metal B might be more important in PARN structural stability. The spectroscopic and aggregation results also suggested that the alterations in the active site structure by metal binding or mutations might lead to a global conformational change of the PARN molecule

Guanine can direct binding specificity of Ru-dppz complexes to DNA through steric effects

X-ray crystal structures of three Λ-[Ru(L)2dppz]2+ complexes (L=phen, bpy) bound to d((5BrC)GGC/GCCG) show the compounds intercalated at a 5´-CG-3´ step. The compounds bind through canted intercalation, with the binding angle determined by the guanine-NH2 group, in contrast to symmetrical intercalation previously observed at 5´-TA-3´ sites. This result suggests that canted intercalation is preferred at 5´-CG-3´ sites even though the site itself is symmetrical, and we hypothesise that symmetrical intercalation in a 5´-CG-3´ step could give rise to a longer luminescence lifetime than canted intercalation

Classification of pseudo pairs between nucleotide bases and amino acids by analysis of nucleotide–protein complexes

Nucleotide bases are recognized by amino acid residues in a variety of DNA/RNA binding and nucleotide binding proteins. In this study, a total of 446 crystal structures of nucleotide–protein complexes are analyzed manually and pseudo pairs together with single and bifurcated hydrogen bonds observed between bases and amino acids are classified and annotated. Only 5 of the 20 usual amino acid residues, Asn, Gln, Asp, Glu and Arg, are able to orient in a coplanar fashion in order to form pseudo pairs with nucleotide bases through two hydrogen bonds. The peptide backbone can also form pseudo pairs with nucleotide bases and presents a strong bias for binding to the adenine base. The Watson–Crick side of the nucleotide bases is the major interaction edge participating in such pseudo pairs. Pseudo pairs between the Watson–Crick edge of guanine and Asp are frequently observed. The Hoogsteen edge of the purine bases is a good discriminatory element in recognition of nucleotide bases by protein side chains through the pseudo pairing: the Hoogsteen edge of adenine is recognized by various amino acids while the Hoogsteen edge of guanine is only recognized by Arg. The sugar edge is rarely recognized by either the side-chain or peptide backbone of amino acid residues

Use of divalent metal ions in the DNA cleavage reaction of topoisomerase IV

It has long been known that type II topoisomerases require divalent metal ions in order to cleave DNA. Kinetic, mutagenesis and structural studies indicate that the eukaryotic enzymes utilize a novel variant of the canonical two-metal-ion mechanism to promote DNA scission. However, the role of metal ions in the cleavage reaction mediated by bacterial type II enzymes has been controversial. Therefore, to resolve this critical issue, this study characterized the DNA cleavage reaction of Escherichia coli topoisomerase IV. We utilized a series of divalent metal ions with varying thiophilicities in conjunction with oligonucleotides that replaced bridging and non-bridging oxygen atoms at (and near) the scissile bond with sulfur atoms. DNA scission was enhanced when thiophilic metal ions were used with substrates that contained bridging sulfur atoms. In addition, the metal-ion dependence of DNA cleavage was sigmoidal in nature, and rates and levels of DNA cleavage increased when metal ion mixtures were used in reactions. Based on these findings, we propose that topoisomerase IV cleaves DNA using a two-metal-ion mechanism in which one of the metal ions makes a critical interaction with the 3′-bridging atom of the scissile phosphate and facilitates DNA scission by the bacterial type II enzyme