Determination of the DNA bend angle induced by the restriction endonuclease EcoRV in the presence of Mg2+.

Stöver, Timo; Köhler, S. Eleonore; Fagin, Ursula; Wende, Wolfgang; Wolfes, Heiner; Pingoud, Alfred

doi:10.1016/s0021-9258(18)52923-9

Cited by 32 publications

(11 citation statements)

References 37 publications

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“…and the EDD triple mutant shows no difference in the gel mobilities of these protein-DNA complexes (data not shown). This result demonstrates that the bending of the DNA is 44°in all four complexes, as determined for the specific D90A-DNA complex (Stover et al, 1993). Therefore, the conformations of all four complexes must be very similar to each other.…”

Section: Resultssupporting

confidence: 62%

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

Maschke²,

Selent³

et al. 1995

Biochemistry

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In contrast to many other type II restriction endonucleases, EcoRV binds specifically to DNA only in the presence Mg2+. According to the co-crystal structure of an EcoRV-DNA complex, Mg2+ ion(s) bind to the active site of EcoRV liganded by Glu45, Asp74, and Asp90. Here we present experimental evidence suggesting that the EcoRV-DNA complex also interacts with Mg2+ ions at other sites: (i) We have prepared an EcoRV triple mutant, in which all acidic amino acids in the catalytic center are replaced by alanine. This mutant is catalytically inactive. It binds nonspecifically to DNA in the absence of Mg2+, whereas it binds specifically to DNA in the presence of Mg2+. This means that Mg2+ induces specific DNA binding in this mutant, although all Mg2+ ligands in the catalytic center are removed. Therefore, additional interactions between Mg2+ and the EcoRV-DNA complex probably occur at sites distinct from the catalytic center. (ii) We have measured the specific and nonspecific DNA binding constants of EcoRV and of the triple mutant in the presence and absence of Mg2+. Mg2+ reduces nonspecific binding by 3-4 orders of magnitude, presumably because Mg2+ ions bound to the DNA have to be released upon complex formation. In contrast, the specific binding of the wild-type enzyme and the triple mutant is increased in the presence of Mg2+. This result can only be explained if a Mg2+ ion binds to the specific EcoRV-DNA complex probably at a site distinct from the catalytic center.(ABSTRACT TRUNCATED AT 250 WORDS)

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Section: Resultssupporting

confidence: 62%

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

Maschke²,

Selent³

et al. 1995

Biochemistry

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“…These contacts contribute to the indirect readout (defined as the contribution of backbone contacts to specific recognition), as opposed to the direct readout (defined as the contribution of base contacts to specific recognition) (review: 34). An unusual property of the DNA in the crystal structure of the specific EcoRV/ DNA complex (9) is its highly distorted conformation characterized by a sharp central kink of approximately 50° ( 9,35,36; Figure 8a). However, this kink originates at the central TA step of the recognition sequence; the rest of the DNA, especially the base pairs flanking the recognition site, is in standard B-conformation (9).…”

Section: Discussionmentioning

confidence: 99%

Engineering of Variants of the Restriction Endonuclease EcoRV That Depend in Their Cleavage Activity on the Flexibility of Sequences Flanking the Recognition Site

1998

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The present work describes mutants of the restriction enzyme EcoRV that discriminate very efficiently between oligodeoxynucleotide substrates with an EcoRV recognition sequence in different sequence context. All of these EcoRV variants harbor substitutions at position 226, where in the cocrystal structure of the specific EcoRV/DNA complex an arginine contacts the backbone of the DNA substrate upstream of the recognition sequence, and cleave an oligodeoxynucleotide with an EcoRV site in a nonflexible sequence context (the recognition site being flanked by runs of A and T) with much higher catalytic efficiency (kcat/Km) than an oligodeoxynucleotide with an EcoRV site in a flexible sequence context (the recognition site being flanked by runs of AT), in contrast to the wild-type enzyme, that cleaves both substrates with the same catalytic efficiency. Steady-state and single-turnover kinetics indicate that the enhanced selectivity of the mutants is due to the catalytic step of the reaction. It is possible to enhance the discriminatory power of these EcoRV variants through the choice of appropriate reaction conditions, in particular low salt concentration and low reaction temperatures. It must be emphasized that the enhanced selectivity of these EcoRV variants toward EcoRV sites in a flexible and nonflexible sequence context, respectively, is not only seen with oligodeoxynucleotides, but also with plasmid substrates.

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“…Thus, the only current routes to a stable and specific ternary complex for EcoRN require either the protein or the metal ion to be altered to prevent catalysis. Moreover, DNA bending angles were determined for D90A with Mg2+ (Stover et al, 1993) and for wild-type EcoRN with Ca2+ (Figure 4). They both yielded angles that were close to the value of 55°seen in the crystal structure (Winkler et al, 1993;Kostrewa & Winkler, 1995).…”

Section: Discussionmentioning

confidence: 99%

“…In the absence of Mg2+, this mutant bound DNA in the same manner as wild-type EcoRV, with no preference for specific over nonspecific sequences, but in the presence of Mg2+, it bound preferentially to the EcoRV site (Thielking et al, 1992) . Moreover, in the specific DNA-protein complex of D90A with Mg2+, the DNA was bent by 44° ( Stover et al, 1993) , an angle similar to that seen in the crystal structure with wild-type EcoRV (Winkler et al" 1993; Kostrewa & Winkler, 1995). However, a potential handicap to this strategy is that the mutation must destroy catalytic activity without destroying DNA-binding activity, and thus it needs to be made at a key residue for catalysis.…”

mentioning

confidence: 81%

Specific DNA Recognition by EcoRV Restriction Endonuclease Induced by Calcium Ions

Vipond¹,

Halford²

1995

Biochemistry

131

136

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In the presence of Mg2+, the EcoRV restriction endonuclease cleaves DNA specifically at its recognition sequence, but in the absence of divalent metal ions, it binds DNA without any specificity: gel-shift experiments had revealed multiple EcoRV-DNA complexes, due to the binding of one, two, three, or more molecules of protein per molecule of DNA, with the same equilibrium constant for each association. In this study, the binding of EcoRV to DNA was measured by gel shift in the presence of Ca2+, an ion that perturbs the Mg(2+)-dependent activity of EcoRV but that fails to support DNA cleavage. With Ca2+, and at a lower concentration of EcoRV protein than that required for binding in the absence of divalent metal ions, a single complex was observed with DNA containing the EcoRV recognition site. This complex was not formed with DNA that had been methylated at the EcoRV site nor with an isogenic DNA lacking the EcoRV recognition site. The single complex thus is due to the specific binding of EcoRV to its recognition site on the DNA. From gel shifts with a permuted set of DNA fragments, the degree of DNA bending by EcoRV at its recognition site was estimated to be 53 degrees +/- 4 degrees. This angle is similar to that seen in the crystal structure of the cognate DNA-protein complex. Calcium ions thus appear to mimic the role of Mg2+ in generating a specific protein-metal-DNA complex, but in contrast to Mg2+, Ca2+ gives a stable ternary complex in which the DNA-bound nuclease cannot cleave the DNA.

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Determination of the DNA bend angle induced by the restriction endonuclease EcoRV in the presence of Mg2+.

Cited by 32 publications

References 37 publications

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

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

Engineering of Variants of the Restriction Endonuclease EcoRV That Depend in Their Cleavage Activity on the Flexibility of Sequences Flanking the Recognition Site

Specific DNA Recognition by EcoRV Restriction Endonuclease Induced by Calcium Ions

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