DNA Binding Specificity of the EcoRV Restriction Endonuclease Is Increased by Mg2+ Binding to a Metal Ion Binding Site Distinct from the Catalytic Center of the Enzyme

Jeltsch, Albert; Maschke, Hans E.; Selent, Ursel; Wenz, Christian; Köhler, S. Eleonore; Connolly, B A; Thorogood, Harry; Pingoud, Alfred

doi:10.1021/bi00018a028

Cited by 56 publications

(62 citation statements)

References 22 publications

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“…It also cannot be ruled out that the lower Mg(II) binding stoichiometries observed for the Glu-9 and Glu-115 mutants might be due to the effect of the mutations on the protein structure and not due to loss of a Mg(II) coordination site. A Mg(II) binding site distinct from the catalytic center has been proposed for the EcoRV restriction endonuclease (32). E. coli DNA topoisomerase I may also have a second Mg(II) binding site away from the active site region that is required for relaxation activity because of its effect on the protein conformational changes that take place during the relaxation reaction cycle (28).…”

Section: Discussionmentioning

confidence: 99%

Site-directed Mutagenesis of Conserved Aspartates, Glutamates and Arginines in the Active Site Region of Escherichia coli DNA Topoisomerase I

Zhu

Roche

Papanicolaou

et al. 1998

Journal of Biological Chemistry

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To catalyze relaxation of supercoiled DNA, DNA topoisomerases form a covalent enzyme-DNA intermediate via nucleophilic attack of a tyrosine hydroxyl group on the DNA phosphodiester backbone bond during the step of DNA cleavage. Strand passage then takes place to change the linking number. This is followed by DNA religation during which the displaced DNA hydroxyl group attacks the phosphotyrosine linkage to reform the DNA phosphodiester bond. Mg(II) is required for the relaxation activity of type IA and type II DNA topoisomerases. A number of conserved amino acids with acidic and basic side chains are present near Tyr-319 in the active site of the crystal structure of the 67-kDa N-terminal fragment of Escherichia coli DNA topoisomerase I. Their roles in enzyme catalysis were investigated by site-directed mutation to alanine. Mutation of Arg-136 abolished all the enzyme relaxation activity even though DNA cleavage activity was retained. The Glu-9, Asp-111, Asp-113, Glu-115, and Arg-321 mutants had partial loss of relaxation activity in vitro. All the mutants failed to complement chromosomal topA mutation in E. coli AS17 at 42°C, possibly accounting for the conservation of these residues in evolution.DNA topoisomerases (for review, see Refs. 1-7) catalyze the interconversion of different DNA topological isomers by first forming a covalent enzyme-DNA intermediate via nucleophilic attack of a tyrosine hydroxyl on the DNA phosphodiester linkage. After strand passage through the break, religation involving nucleophilic attack of the displaced DNA hydroxyl group on the phosphotyrosine linkage takes place. Type IA and type II DNA topoisomerases are linked to the 5Ј-phosphoryl end of the cleaved DNA while type IB DNA topoisomerases are linked to the 3Ј-phosphoryl end. Mg(II) is required for the relaxation activities of both type IA and type II DNA topoisomerases but not for the type IB enzymes. The detailed catalytic mechanism of DNA cleavage and religation by topoisomerases remains to be elucidated. The mechanism of the type IA and type II topoisomerase may share similarities with other enzymes that also require Mg(II) for nucleotidyl transfer activity.Tyr-319 of Escherichia coli DNA topoisomerase I is the catalytic residue that provides the hydroxyl group for forming the covalent intermediate with DNA. The three-dimensional structure of the 67-kDa N-terminal domain of this enzyme has been determined by x-ray crystallography (8). In this structure, Tyr-319 is present in the interface between domains I and III. It has been pointed out (8) that the spatial arrangement of the three acidic residues Asp-111, Asp-113, and Glu-115 in the active site region is similar to the acidic residues that coordinate two divalent cations in the exonuclease catalytic site of Klenow fragment (9). However, the structure observed has to undergo additional conformational changes before there is sufficient space in the active site region for DNA and possibly Mg(II) to bind. A number of residues found in the active site, including Glu-9, Asp-111,...

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Section: Discussionmentioning

confidence: 99%

Site-directed Mutagenesis of Conserved Aspartates, Glutamates and Arginines in the Active Site Region of Escherichia coli DNA Topoisomerase I

Zhu

Roche

Papanicolaou

et al. 1998

Journal of Biological Chemistry

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“…M13 DNA, containing R p -phosphorothioates, showed that GATsATC sequences were very refractory to hydrolysis and substitution elsewhere tended to reduce cutting rates (32). Very recently, the phosphorothioate oligonucleotides used in this study have been used to show the presence of a metal ion binding site distinct from the catalytic center (55). In this publication, we examine the effects of both isomers of phosphorothioates within and immediately flanking the EcoRV restriction site on endonuclease-catalyzed hydrolysis and relate the results found to the available crystal structures.…”

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confidence: 88%

“…Two further points deserve mentioning. First, by comparing the Mg 2ϩ -and Mn 2ϩ -catalyzed cleavage of the phosphorothioates, an additional metal ion, remote from the active site, was proposed to bind to the GACGpATATCGTC phosphate (55). The exact role of this metal ion in selectivity remains obscure, but it could certainly be incorporated as an additional structural element in the network illustrated.…”

Section: Figmentioning

confidence: 99%

Influence of the Phosphate Backbone on the Recognition and Hydrolysis of DNA by the EcoRV Restriction Endonuclease

Thorogood

Grasby

Connolly

1996

Journal of Biological Chemistry

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“…1a), a 20-mer oligonucleotide substrate was chosen for a detailed kinetic analysis of the mutant proteins under steadystate conditions. This substrate can be expected to mimic a high molecular weight DNA as it is longer than the entire binding pocket of EcoRV, which covers 14 -16 bp (5,20). By variation of the substrate concentration over 5 orders of magnitude, it was possible to determine the kinetic parameters K m and k cat for all mutants except the T37A mutant (Table III).…”

Section: Selection Generation and Purification Of Ecorv Mutants-mentioning

confidence: 99%

Probing the Indirect Readout of the Restriction Enzyme EcoRV

Wenz

Jeltsch

Pingoud

1996

Journal of Biological Chemistry

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According to the crystal structure of the specific EcoRV⅐DNA complex, not only the functional groups of the nucleobases but also the phosphate groups of the DNA backbone are contacted by the enzyme. To examine the contribution of backbone contacts to substrate recognition and catalysis by EcoRV, we exchanged 12 amino acids residues located close to phosphate groups by site-directed mutagenesis. We purified the resulting EcoRV mutants and characterized them with respect to their DNA binding and cleavage activity. According to our steady state kinetic analysis, there are strong interactions between three basic amino acid residues (Lys-119, Arg-140, and Arg-226) and the phosphate backbone that support specific binding presumably by inducing and maintaining the kinked conformation of the DNA observed in the specific EcoRV⅐DNA complex. These contacts are important in both the ground state and the transition state. Other, uncharged residues (Thr-93 and Ser-112), which could be involved in hydrogen bonds to the phosphate groups, are needed primarily to stabilize the transition state. An especially important amino acid residue is Thr-37, which seems to couple recognition to catalysis by indirect readout.The specific interaction of proteins with DNA is of fundamental importance in biology. Much effort, therefore, has been undertaken to understand the molecular basis of the underlying recognition process. It has been suggested that specificity is due to specific hydrogen bonds between the protein and the edges of the bases of the DNA (direct readout) (1). Such contacts are indeed observed in nearly every structure of specific protein-DNA complexes determined so far. In the first co-crystal structure of the trp repressor and its operator DNA, however, no direct contacts to the bases were seen that could account for the observed specificity of DNA binding; it was argued, therefore, that in this case, specific recognition of DNA is primarily due to contacts to the phosphate groups of the DNA backbone (indirect readout) (2). This means that during interaction with DNA, the protein recognizes a specific sequencedependent conformation of the phosphodiester backbone in addition to functional groups of the bases. This concept was initially met with some reservation, but it has proven to be very fruitful, although it was recently demonstrated that the specific interaction between the trp repressor and its operator is not only due to phosphate contacts but also due to watermediated base contacts as well as DNA-induced tetramerization of the protein on the DNA (3). From comparisons of cocrystal structures of specific and nonspecific complexes of DNA binding proteins with DNA, it became evident that specific recognition of DNA by proteins is mediated by direct and indirect readout also in other systems (e.g. glucocorticoid receptor (4); EcoRV (5)); in general, in the specific complexes, much more interactions are observed between the protein and the bases but also between the protein and the phosphate backbone of the DNA than in the ...

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DNA Binding Specificity of the EcoRV Restriction Endonuclease Is Increased by Mg2+ Binding to a Metal Ion Binding Site Distinct from the Catalytic Center of the Enzyme

Cited by 56 publications

References 22 publications

Site-directed Mutagenesis of Conserved Aspartates, Glutamates and Arginines in the Active Site Region of Escherichia coli DNA Topoisomerase I

Site-directed Mutagenesis of Conserved Aspartates, Glutamates and Arginines in the Active Site Region of Escherichia coli DNA Topoisomerase I

Influence of the Phosphate Backbone on the Recognition and Hydrolysis of DNA by the EcoRV Restriction Endonuclease

Probing the Indirect Readout of the Restriction Enzyme EcoRV

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