Imaging DNA Loops Induced by Restriction Endonuclease EcoRII

Mücke, Merlind; Mackeldanz, Petra; Behlke, Joachim; Krüger, Detlev H.; Reuter, Monika

doi:10.1074/jbc.m003904200

Cited by 29 publications

(10 citation statements)

References 35 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…However, in the presence of Ca 2ϩ ions, EcoRII has been reported to bind to the target DNA but not cleave the DNA strand (10). In fact, transmission electron microscopy has confirmed the formation of the Eco-RII/DNA complex in the presence of Ca 2ϩ ions (10).…”

Section: Binding Behavior Of Ecorii In the Presence Of Camentioning

confidence: 82%

“…It recognizes two units of recognition sequences (5Ј-CCWGG-3Ј) included in one DNA chain (cis-binding) or in two DNA chains one by one (trans-binding), and cleaves either site (see Fig. 1B) (7)(8)(9)(10). Therefore, it is difficult to distinguish between cis and trans binding in the bulk solution by conventional analysis methods such as electrophoresis mobility shift assay and fluorescent anisotropy.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Direct Monitoring of Allosteric Recognition of Type IIE Restriction Endonuclease EcoRII

Takahashi

Matsuno

Furusawa

et al. 2008

Journal of Biological Chemistry

View full text Add to dashboard Cite

EcoRII is a homodimer with two domains consisting of a DNA-binding N terminus and a catalytic C terminus and recognizes two specific sequences on DNA. It shows a relatively complicated cleavage reaction in bulk solution. After binding to either recognition site, EcoRII cleaves the other recognition site of the same DNA (cis-binding) strand and/or the recognition site of the other DNA (trans-binding) strand. Although it is difficult to separate these two reactions in bulk solution, we could simply obtain the binding and cleavage kinetics of only the cisbinding by following the frequency (mass) changes of a DNAimmobilized quartz-crystal microbalance (QCM) responding to the addition of EcoRII in aqueous solution. We obtained the maximum binding amounts (⌬m max ), the dissociation constants (K d ), the binding and dissociation rate constants (k on and k off ), and the catalytic cleavage reaction rate constants (k cat ) for wildtype EcoRII, the N-terminal-truncated form (EcoRII N-domain), and the mutant derivatives in its C-terminal domain (K263A and R330A). It was determined from the kinetic analyses that the N-domain, which covers the catalytic C-domain in the absence of DNA, preferentially binds to the one DNA recognition site while transforming EcoRII into an active form allosterically, and then the secondary C-domain binds to and cleaves the other recognition site of the DNA strand. DNA-binding proteins, which specifically recognize DNA sequences, play an important role in maintaining and managing genetic information in all living cells. To study the binding mechanisms of these proteins to DNA is important not only for a biological understanding of gene expression but also for the design of an artificial DNA-binding molecule. A restriction endonuclease is also a specific DNA-binding protein. Bacteria possess restriction endonucleases to protect them from infection by digesting the foreign DNA from viruses. In addition to being an essential tool for gene manipulation in the genetic engineering field, they have been widely used as tools to understand DNA recognition and cleavage mechanisms.Thousands of restriction endonucleases have been discovered thus far, and classified into several types because of their cleavage reactions shown in Fig. 1A (1-3). Type IIP restriction endonucleases, which are commonly used in the genetic engineering field, recognize simply one palindromic sequence (4 -8 base pairs), which is cleaved. On the other hand, Type IIE or IIF endonucleases need two recognition sites and cleave one or two sites, respectively (4). EcoRII, which is classified as a type IIE restriction endonuclease, is a homodimer (5, 6). It recognizes two units of recognition sequences (5Ј-CCWGG-3Ј) included in one DNA chain (cis-binding) or in two DNA chains one by one (trans-binding), and cleaves either site (see Fig. 1B) (7-10). Therefore, it is difficult to distinguish between cis and trans binding in the bulk solution by conventional analysis methods such as electrophoresis mobility shift assay and fluorescent anisot...

show abstract

Section: Binding Behavior Of Ecorii In the Presence Of Camentioning

confidence: 82%

mentioning

confidence: 99%

Direct Monitoring of Allosteric Recognition of Type IIE Restriction Endonuclease EcoRII

Takahashi

Matsuno

Furusawa

et al. 2008

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…Mutations in the C-terminal domain that inactivated the catalytic center but rendered the DNA recognition function of the C-terminal domain largely unaffected turned Sau3AI into a type IIe enzyme. As a matter of fact, Sau3AI resembles the EcoRII V258N variant, which (due to the amino acid substitution) is monomeric, but can dimerize in the presence of DNA, albeit at lower efficiency, and presumably therefore has only little activity (58). It is noteworthy that the type IIe enzyme NaeI is the outcome of a different evolutionary scenario: as shown by the crystal structure analysis, NaeI is composed of two very different domains, an N-terminal catalytic domain (which is also responsible for dimerization) with a typical restriction enzyme fold and a Cterminal domain with a strong structural resemblance to the catabolite activator protein, which functions as the effector domain (12).…”

Section: Discussionmentioning

confidence: 99%

Sau3AI, a Monomeric Type II Restriction Endonuclease That Dimerizes on the DNA and Thereby Induces DNA Loops

Friedhoff

Lurz

Lüder

et al. 2001

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Here, we report that Sau3AI, an unusually large type II restriction enzyme with sequence homology to the mismatch repair protein MutH, is a monomeric enzyme as shown by gel filtration and ultracentrifugation. Structural similarities in the N-and C-terminal halves of the protein suggest that Sau3AI is a pseudo-dimer, i.e. a polypeptide with two similar domains. Since Sau3AI displays a nonlinear dependence of cleavage activity on enzyme concentration and a strong preference for substrates with two recognition sites over those with only one, it is likely that the functionally active form of Sau3AI is a dimer of a pseudo-dimer. Indeed, electron microscopy studies demonstrate that two distant recognition sites are brought together through DNA looping induced by the simultaneous binding of two Sau3AI molecules to the DNA. We suggest that the dimeric form of Sau3AI supplies two DNA-binding sites, one that is associated with the catalytic center and one that serves as an effector site.

show abstract

“…Evidence in the literature for two-site behavior of REases comes from a variety of studies. In several cases looped complexes have been directly imaged by electron microscopy (NaeI, Cfr10I, EcoRII, and Sau3AI) (38)(39)(40)(41). DNA looping by SfiI and Cfr10I has been inferred in DNA recombination and gel mobility-shift measurements (42)(43)(44), by NgoMIV in FRET measurements (45), by BspMI in magnetic tweezers experiments (46), and by NarI in tethered particle experiments (47).…”

Section: Methodsmentioning

confidence: 99%

Tension-dependent DNA cleavage by restriction endonucleases: Two-site enzymes are “switched off” at low force

Gemmen

Millin

Smith

2006

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

DNA looping occurs in many important protein-DNA interactions, including those regulating replication, transcription, and recombination. Recent theoretical studies predict that tension of only a few piconewtons acting on DNA would almost completely inhibit DNA looping. Here, we study restriction endonucleases that require interaction at two separated sites for efficient cleavage. Using optical tweezers we measured the dependence of cleavage activity on DNA tension with 15 known or suspected two-site enzymes (BfiI, BpmI, BsgI, BspMI, Cfr9I, Cfr10I, Eco57I, EcoRII, FokI, HpaII, MboII, NarI, SacII, Sau3AI, and SgrAI) and six one-site enzymes (BamHI, EcoRI, EcoRV, HaeIII, HindIII, and DNaseI). All of the one-site enzymes were virtually unaffected by 5 pN of tension, whereas all of the two-site enzymes were completely inhibited. These enzymes thus constitute a remarkable example of a tension sensing ''molecular switch.'' A detailed study of one enzyme, Sau3AI, indicated that the activity decreased exponentially with tension and the decrease was Ϸ10-fold at 0.7 pN. At higher forces (Ϸ20 -40 pN) cleavage by the one-site enzymes EcoRV and HaeIII was partly inhibited and cleavage by HindIII was enhanced, whereas BamHI, EcoRI, and DNaseI were largely unaffected. These findings correlate with structural data showing that EcoRV bends DNA sharply, whereas BamHI, EcoRI, and DNaseI do not. Thus, DNA-directed enzyme activity involving either DNA looping or bending can be modulated by tension, a mechanism that could facilitate mechanosensory transduction in vivo.DNA looping ͉ protein-DNA interactions ͉ single-molecule manipulation R estriction endonucleases (REases) are prokaryotic enzymes that act to ''restrict'' invasion of foreign DNA by cleaving phosphodiester bonds (1). These enzymes also serve as indispensable tools in molecular biology research and are used in procedures such as DNA cloning, fingerprinting, mapping, and sequencing (2). From the perspective of molecular biophysics, these enzymes are excellent model systems for studying basic principles of protein-DNA interactions (3, 4).The most commonly studied REases are of the type II variety, which cleave within or near specific recognition sites, usually require Mg 2ϩ ions as a cofactor, and do not hydrolyze ATP. More than 3,500 different type II REases having Ͼ200 different binding specificities have been identified (5). Of particular interest in our present study are the many unorthodox type II REases that do not cleave DNA efficiently if the template contains only one recognition site (6, 7). Efficient cleavage is only observed with templates containing two or more sites, suggesting that the active complex binds at two sites and the intervening DNA is looped out (7). This phenomenon of DNA looping is of broad importance in molecular biology and plays a role in many key processes including DNA transcription, replication, recombination, and repair (8-13). Looping of DNA by the Lac and Gal repressor proteins in Escherichia coli, for example, is well demonstrated and h...

show abstract

Imaging DNA Loops Induced by Restriction Endonuclease EcoRII

Cited by 29 publications

References 35 publications

Direct Monitoring of Allosteric Recognition of Type IIE Restriction Endonuclease EcoRII

Direct Monitoring of Allosteric Recognition of Type IIE Restriction Endonuclease EcoRII

Sau3AI, a Monomeric Type II Restriction Endonuclease That Dimerizes on the DNA and Thereby Induces DNA Loops

Tension-dependent DNA cleavage by restriction endonucleases: Two-site enzymes are “switched off” at low force

Contact Info

Product

Resources

About