Mechanism of the interaction of <i>Eco</i>RII restriction endonuclease with two recognition sites

Petrauskene, Olga V.; Кубарева, Е. А.; Gromova, Elisaveta S.; Shabarova, Zoe A.

doi:10.1111/j.1432-1033.1992.tb17226.x

Cited by 17 publications

(14 citation statements)

References 16 publications

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“…19), which being somewhat larger than SsoII is a dimer of identical subunits each consisting of 404 amino acid residues (20,21). As a type IIE enzyme EcoRII has two DNA-binding sites, one associated with the catalytic center and the other serving as an effector site (22) (which has led to the acronym type IIE) (23)(24)(25), similar to that shown for NaeI (26 -29). The recognition sequence of EcoRII is 2CCWGG (30), which means that SsoII and EcoRII can be considered to be quasi isoschizomers.…”

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confidence: 89%

Evolutionary Relationship between Different Subgroups of Restriction Endonucleases

Pingoud¹,

Кубарева²,

Stengel³

et al. 2002

Journal of Biological Chemistry

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The type II restriction endonuclease SsoII shows sequence similarity with 10 other restriction endonucleases, among them the type IIE restriction endonuclease EcoRII, which requires binding to an effector site for efficient DNA cleavage, and the type IIF restriction endonuclease NgoMIV, which is active as a homotetramer and cleaves DNA with two recognition sites in a concerted reaction. We show here that SsoII is an orthodox type II enzyme, which is active as a homodimer and does not require activation by binding to an effector site. Nevertheless, it shares with EcoRII and NgoMIV a very similar DNA-binding site and catalytic center as shown here by a mutational analysis, indicative of an evolutionary relationship between these three enzymes. We suggest that a similar relationship exists between other orthodox type II, type IIE, and type IIF restriction endonucleases. This may explain why similarities may be more pronounced between members of different subtypes of restriction enzymes than among the members of a given subtype.More than 3000 different type II restriction endonucleases are known and characterized with respect to their cleavage specificities (1). This group of enzymes probably constitutes one of the largest families of enzymes with the same basic function. This makes type II restriction endonucleases (like DNA methyltransferases) ideal objects to study evolutionary relationships. Moreover, in principle, the family relationships should eventually allow predicting structural and functional features of an individual restriction endonuclease, solely on the basis of sequence comparisons. This was not obvious for a long time since only 10 years ago restriction endonucleases, because of their little sequence conservation, were not considered to be related in evolution (2, 3). But this changed due to the progress made in the analysis of sequence similarities (4 -6) and, in particular, due to the increasing number of crystal structures of restriction enzymes (reviewed in Refs. 7 and 8), which made it obvious that these enzymes have very similar structural cores. Nevertheless, it has been shown recently that some genuine restriction enzymes belong to distinct nuclease superfamilies (Nuc, HNH, and GIY-YIG), which are unrelated and structurally dissimilar to each other and to the "archetypal" (P)D . . . (D/E)XK superfamily (9 -11). These findings make comparative studies using sequences of restriction enzymes even more challenging.We have begun to study the restriction endonuclease SsoII recently (12-15). It is a type II enzyme (reviewed in Refs. 8 and 16) composed of identical subunits each consisting of 305 amino acid residues (17). It cleaves the palindromic sequence 2CC-NGG in the presence of Mg 2ϩ as indicated (18). Our interest in studying this enzyme is due to its sequence similarity to Eco-RII, a type IIE enzyme (reviewed in Ref. 19), which being somewhat larger than SsoII is a dimer of identical subunits each consisting of 404 amino acid residues (20, 21). As a type IIE enzyme EcoRII has two DNA-binding...

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mentioning

confidence: 89%

Evolutionary Relationship between Different Subgroups of Restriction Endonucleases

Pingoud¹,

Кубарева²,

Stengel³

et al. 2002

Journal of Biological Chemistry

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“…This resistance can be also overcome by the presence of modi® ed EcoRII sites, which are usually resistant or almost resistant to EcoRII endonuclease cleavage. However, these modi® ed sites can also be cleaved when they are used as activators for remote recognition sites (6). To understand the role of DNA activator in the cleavage reaction and to elucidate the complex formation process, we have investigated with native (nondenaturing) gel electrophoresis the binding of EcoRII to short DNA-duplexe s containing nucleotide replacements in the recognition site.…”

Section: Introductionmentioning

confidence: 99%

A Model of EcoRII Restriction Endonuclease Action: The Active Complex is Most Likely Formed by One Protein Subunit and One DNA Recognition Site

1999

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SummaryTo elucidate the mechanism of interaction of restriction endonuclease EcoRII with DNA, we studied by native gel electrophoresis the binding of this endonuclease to a set of synthetic DNAduplexescontaining the modi® ed or canonical recognition sequence Keywords EcoRII restriction endonuclease; enzyme±substrate complexes; mechanism of enzyme action; modi® ed DNA substrates; nondenaturing polyacrylamide gel assay.

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“…For example, Type IIE endonucleases such as NaeI and EcoRII are homodimeric proteins that have two distinct DNA binding clefts (18 -20) and thus are capable of binding two copies of their recognition sequence per protein dimer. However, only one of these recognition sequences is cleaved while the other serves as an allosteric effector (21)(22)(23)(24)(25)(26)(27)(28). Studies with NaeI reveal that the recognition sequences flanked by G/C-rich regions are preferred by the activator site, whereas the catalytic site prefers A/T-rich regions (22,25).…”

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Kinetic Analysis of the Coordinated Interaction of SgrAI Restriction Endonuclease with Different DNA Targets

Hingorani-Varma

Bitinaite

2003

Journal of Biological Chemistry

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SgrAI restriction endonuclease cooperatively interacts and cleaves two target sites that include both the canonical sites, CPuCCGGPyG, and the secondary sites, CPuCCGGPy(A/T/C). It has been observed that the cleaved canonical sites stimulate SgrAI cleavage at the secondary sites. Equilibrium binding studies show that SgrAI binds to its canonical sites with a high affinity (K a ‫؍‬ 4 -8 ؋ 10 10 M ؊1 ) and that it has a 15-fold lower affinity for the cleaved canonical sites and a 30-fold lower affinity for the secondary sites. Steady-state kinetics reveals substrate cooperativity for SgrAI cleavage on both canonical and secondary sites. The specificity of SgrAI for the secondary site CACCGGCT, as measured by k cat /K is about 500-fold lower than that for the canonical site CACCGGCG, but this difference is reduced to 10-fold in the presence of the cleaved canonical sites. The efficiency of canonical site cleavage also increases by 3-fold when the cleaved canonical sites are present in the reaction. Furthermore, the substrate cooperativity for SgrAI cleavage is abolished for both types of sites in the presence of cleaved canonical sites. These results indicate that target site cleavage occurs via a coordinated interaction of two SgrAI protein subunits, where the subunit bound to the cleaved site stimulates the cleavage of the uncut site bound by the other subunit. The free subunits of SgrAI have the flexibility to bind different target sites and, consequently, assemble into various catalytically active complexes, which differ in their catalytic efficiencies.Restriction endonucleases serve as useful models to study the molecular basis of specificity in the interaction of proteins with DNA. The prototypical homodimeric Type IIP endonuclease recognizes a symmetrical palindromic sequence, 4 -8 bp long and in the presence of Mg 2ϩ specifically cleaves within the recognition site (1, 2). These enzymes, including EcoRI, EcoRV, BamHI, and PvuII are highly sequence-specific with a very strong ability to discriminate between specific and nonspecific sites, both at the level of DNA binding (recognition) as well as DNA cleavage (catalysis) (3-10). For example, the equilibriumassociation constant of EcoRV for specific DNA is 0.2-4 ϫ 10 10

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Mechanism of the interaction of EcoRII restriction endonuclease with two recognition sites

Cited by 17 publications

References 16 publications

Evolutionary Relationship between Different Subgroups of Restriction Endonucleases

Evolutionary Relationship between Different Subgroups of Restriction Endonucleases

A Model of EcoRII Restriction Endonuclease Action: The Active Complex is Most Likely Formed by One Protein Subunit and One DNA Recognition Site

Kinetic Analysis of the Coordinated Interaction of SgrAI Restriction Endonuclease with Different DNA Targets

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