DNA Binding and Recognition by the IIs Restriction Endonuclease MboII

Soundararajan, M.; Chang, Zhiyuh; Morgan, Richard D.; Heslop, Pauline; Connolly, Bernard A.

doi:10.1074/jbc.m109100200

Cited by 31 publications

(31 citation statements)

References 46 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…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). Evidence in other cases comes from the comparison of DNA cleavage rates on templates containing two sites versus only one site, which has been reported for BfiI, BsgI, BpmI, FokI, MboII, NarI, and SgrAI (26,28,48,49). Finally, stimulation of activity upon addition of short oligonucleotide duplexes containing the recognition sequence has been reported for Eco57I, HpaII, Cfr9I, and SacII.…”

Section: Methodsmentioning

confidence: 98%

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

Section: Methodsmentioning

confidence: 98%

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

show abstract

“…Moreover, although some of the type II restriction enzymes that recognize palindromic DNA sequences have to interact with two copies of the relevant recognition sequence before they can cleave DNA (11,12), the requirement for two sites seems to be particularly common, although not universal, among the type IIs enzymes. Other type IIs enzymes that have recently been shown to need two sites include MboII (48), which follows scheme B, like BsgI (Fig. 3), and BfiI, 4 that yields reaction profiles similar to those for scheme C, like FokI (Fig.…”

Section: Discussionmentioning

confidence: 99%

Many Type IIs Restriction Endonucleases Interact with Two Recognition Sites before Cleaving DNA

Bath

Milsom

Gormley

et al. 2002

Journal of Biological Chemistry

114

168

View full text Add to dashboard Cite

Type IIs restriction endonucleases recognize asymmetric DNA sequences and cleave both DNA strands at fixed positions, typically several base pairs away from the recognition site. These enzymes are generally monomers that transiently associate to form dimers to cleave both strands. Their reactions could involve bridging interactions between two copies of their recognition sequence. To examine this possibility, several type IIs enzymes were tested against substrates with either one or two target sites. Some of the enzymes cleaved the DNA with two target sites at the same rate as that with one site, but most cut their two-site substrate more rapidly than the one-site DNA. In some cases, the two sites were cut sequentially, at rates that were equal to each other but that exceeded the rate on the onesite DNA. In another case, the DNA with two sites was cleaved rapidly at one site, but the residual site was cleaved at a much slower rate. In a further example, the two sites were cleaved concertedly to give directly the final products cut at both sites. Many type IIs enzymes thus interact with two copies of their recognition sequence before cleaving DNA, although via several different mechanisms.Over 3000 type II restriction endonucleases have been identified to date (1). These enzymes recognize short DNA sequences, 4 -8 bp long and cleave both strands of the DNA at fixed locations in or near their recognition sites (2). With one exception, BfiI (3), they require Mg 2ϩ or a similar divalent metal ion to cleave DNA (4), although a few also require AdoMet 1 for maximal activity (5). Many of the type II enzymes are homodimeric proteins that interact symmetrically with palindromic DNA sequences, so that one active site in the dimer is placed to cleave one strand of the DNA and the other active site the equivalent phosphodiester bond in the opposite strand: for example, EcoRV, BamHI, and BglI (6 -8). Enzymes of this sort cleave DNA with multiple target sites by means of separate reactions at each site (9), although they can act processively and migrate from one site to another by intramolecular processes (10).Several type II enzymes that recognize palindromic sequences differ from the orthodox enzymes, because they have to interact with two copies of their target sequence before they can cleave DNA (11,12). The latter include the type IIe enzymes, such as EcoRII, NaeI, and Sau3AI (13-17), and the type IIf enzymes, such as SfiI, SgrAI, Cfr10I,. Both the type IIe and IIf enzymes bind two sites concurrently but the former cleave only one site per turnover, whereas the latter cleave both sites concertedly within a single turnover (22). Except for Sau3AI, a monomer in free solution (17), the type IIe enzymes are dimers with two distinct DNA-binding clefts, both of which bind the cognate DNA sequence but one is an allosteric locus that activates DNA cleavage in the other cleft (15,16). In contrast, the type IIf enzymes are generally tetramers with two identical surfaces for binding their palindromic sites, each made from two subun...

show abstract

“…As is common for type IIS restriction enzymes (10,11), BfiI cleaves plasmids with two copies of its recognition sequence more rapidly than plasmids with one site (24). The BfiI dimer also forms synaptic complexes with two cognate DNA molecules (24).…”

Section: A Hairpin Intermediate?mentioning

confidence: 99%

“…To make doublestrand breaks, the monomer of FokI bound to its recognition site associates transiently with a second monomer to form a dimer with two active sites (7-9). Many type IIS enzymes operate in this way (10,11). …”

mentioning

confidence: 99%

How the Bfi I restriction enzyme uses one active site to cut two DNA strands

Sasnauskas

Halford

Šikšnys

2003

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

View full text Add to dashboard Cite

Unlike other restriction enzymes, BfiI functions without metal ions. It recognizes an asymmetric DNA sequence, 5 -ACTGGG-3 , and cuts top and bottom strands at fixed positions downstream of this sequence. Many restriction enzymes are dimers of identical subunits, with one active site for each DNA strand. Others, like FokI, dimerize transiently during catalysis. BfiI is also a dimer but it has only one active site, at the dimer interface. We show here that BfiI remains a dimer as it makes double-strand breaks in DNA and that its single active site acts sequentially, first on the bottom and then the top strand. Hence, after cutting the bottom strand, a rearrangement of either the protein and͞or the DNA in the BfiI-DNA complex must switch the active site to the top strand. Low pH values selectively block top-strand cleavage, converting BfiI into a nicking enzyme that cleaves only the bottom strand. The switch to the top strand may depend on the ionization of the cleaved 5 phosphate in the bottom strand. BfiI thus uses a mechanism for making double-strand breaks that is novel among restriction enzymes.S equence-specific cleavage of double-stranded DNA underpins many genetic events, including recombination, transposition, viral DNA integration, and the restriction of DNA (1-4). The enzymes that make double-strand breaks use many different strategies to achieve this end. The simplest may be the orthodox type II restriction enzymes such as EcoRI or EcoRV, dimers of identical subunits that recognize palindromic DNA sequences. In the presence of Mg 2ϩ , they cleave both strands at fixed positions in their recognition sites (3). They bind their target sites symmetrically, so that one active site from the dimer is positioned against one DNA strand and likewise the second active site on the other strand. Independent reactions in each active site then generate the double-strand break. Some homing endonucleases, such as I-CreI, also act in this way (4), as do the enzymes that resolve Holliday junctions (2).This strategy cannot apply to enzymes that recognize nonpalindromic sites and cut both strands away from the recognition site (3). For example, FokI, a type IIS restriction enzyme, recognizes the sequence 5Ј-GGATG-3Ј and cuts top and bottom strands 9 and 13 nt downstream of this site, respectively (5). FokI is a monomer with separate domains for DNA recognition and catalysis. The catalytic domain has a single active site, which is like that in each subunit of an orthodox enzyme, so the FokI monomer cannot cleave both DNA strands (6). To make doublestrand breaks, the monomer of FokI bound to its recognition site associates transiently with a second monomer to form a dimer with two active sites (7-9). Many type IIS enzymes operate in this way (10,11).An alternative to using a homodimeric enzyme to make double-strand breaks is an enzyme with different subunits. For example, the transposition of Tn7 requires two proteins, TnsA and TnsB, that each cleave one strand of the DNA (12). Interestingly, the structure of TnsA is similar...

show abstract

DNA Binding and Recognition by the IIs Restriction Endonuclease MboII

Cited by 31 publications

References 46 publications

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

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

Many Type IIs Restriction Endonucleases Interact with Two Recognition Sites before Cleaving DNA

How the Bfi I restriction enzyme uses one active site to cut two DNA strands

Contact Info

Product

Resources

About