BfiI, a restriction endonuclease from Bacillus firmus S8120, which recognizes the novel non-palindromic sequence 5'-ACTGGG(N)5/4-3'

Vitkute, Jolanta; Manelienė, Z.; Petrušyté, M.; Janulaitis, A.

doi:10.1093/nar/26.14.3348

Cited by 18 publications

(21 citation statements)

References 8 publications

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“…In addition to the previously reported site of action of BfiI on the top strand, 5 nt downstream of the recognition sequence (23), additional cleavages of the top strand were also detected 6 and 7 nt away from the site: they constituted 30% and 10% of the reaction products, respectively (data not shown). The same pattern of top-strand cleavage was observed with two other 30-bp duplexes that have the recognition sequence for BfiI flanked by different sequences, but they were all cleaved in the bottom strand only at the cognate position 4 nt away.…”

Section: Resultssupporting

confidence: 58%

“…BfiI must therefore hydrolyze phosphodiester bonds by a radically different mechanism from all restriction enzymes characterized to date (22). Like FokI (5), BfiI is a type IIS endonuclease: it recognizes an asymmetric sequence, ACTGGG and cleaves top and bottom strands 5 and 4 nt downstream of this site (23). Sequence alignments and site-directed mutagenesis (22,24) indicate that the N-terminal half of BfiI is similar to an EDTA-resistant DNase that lacks sequence specificity, Nuc of Salmonella typhimurium (25).…”

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confidence: 99%

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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.

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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...

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Section: Resultssupporting

confidence: 58%

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.

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“…BfiI cleaves DNA at specified positions downstream from an asymmetric recognition sequence (11). Unlike other REases, it functions without metal ions (12).…”

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confidence: 99%

Site-specific DNA transesterification catalyzed by a restriction enzyme

Sasnauskas¹,

Connolly

Halford

et al. 2007

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

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Most restriction endonucleases use Mg 2؉ to hydrolyze phosphodiester bonds at specific DNA sites. We show here that BfiI, a metal-independent restriction enzyme from the phospholipase D superfamily, catalyzes both DNA hydrolysis and transesterification reactions at its recognition site. In the presence of alcohols such as ethanol or glycerol, it attaches the alcohol covalently to the 5 terminus of the cleaved DNA. Under certain conditions, the terminal 3 -OH of one DNA strand can attack the target phosphodiester bond in the other strand to create a DNA hairpin. Transesterification reactions on DNA with phosphorothioate linkages at the target bond proceed with retention of stereoconfiguration at the phosphorus, indicating, uniquely for a restriction enzyme, a twostep mechanism. We propose that BfiI first makes a covalent enzyme-DNA intermediate, and then it resolves it by a nucleophilic attack of water or an alcohol, to yield hydrolysis or transesterification products, respectively.I n the presence of Mg 2ϩ , type II restriction endonucleases (REases) hydrolyze specific phosphodiester bonds in DNA to yield 5Ј-phosphate and 3Ј-hydroxyl termini (1). The stereochemical pathway for phosphodiester hydrolysis by four such enzymes, EcoRI, EcoRV, SfiI, and HpaII (2-4), have been determined. All proceed with inversion of configuration at the scissile phosphate, which implies an odd number of chemical steps in the hydrolytic reaction, and it is most simply accounted for by a direct in-line displacement of the 3Ј-leaving group by water (5-7). High-resolution structures are available for many more REases than have been analyzed stereochemically (1). Their active sites are generally similar, with several carboxylates to act as ligands for Mg 2ϩ in spatially equivalent positions (1). All such enzymes probably act in the same way, by using water to attack the scissile phosphate, to invert its configuration.Endonucleases from the transposase-retroviral integrase family, like MuA or HIV-integrase, catalyze at a single active site two sets of reactions on DNA (4, 8, 9). The first, the endonucleolytic cleavage of the 3Ј end of the transposon or viral DNA, is mechanistically similar to the hydrolysis reactions of REases. The second, termed DNA strand transfer, is a single-step transesterification in which the newly liberated 3Ј-OH group attacks and becomes linked to a phosphate group in the target DNA (9, 10).We ask here whether the BfiI REase can catalyze transesterification reactions in addition to DNA hydrolysis. BfiI cleaves DNA at specified positions downstream from an asymmetric recognition sequence (11). Unlike other REases, it functions without metal ions (12). The N-terminal half of BfiI (13, 14) is similar to Nuc, an EDTA-resistant nuclease from Salmonella typhimurium (15, 16) that belongs to the phospholipase D (PLD) superfamily. The PLD superfamily is a diverse group of proteins that includes phospholipases, phospholipid synthases, bacterial toxins, phosphodiesterases (PDEs), and nucleases (17, 18). The PLD enzymes catal...

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“…Four rounds of chromosomal walking by inverse PCR and sequencing identified bmrIM2 and bmrIR coding sequences adjacent to the M1 genes. The bmrIM1M2 genes and bmrIR gene are transcribed in opposite directions, analogous to the BfiI R-M system ( Figure 1A) [19]. The BmrI and BfiI REases are highly homologous, sharing 78% amino acid sequence identity and 89% amino acid sequence similarity ( Figure 1B).…”

Section: Cloning and Expression Of The Bmri R-m Systemmentioning

confidence: 99%

Expression and purification of BmrI restriction endonuclease and its N-terminal cleavage domain variants

Bao

Higgins

Zhang

et al. 2008

Protein Expression and Purification

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BmrI (ACTGGG N 5 /N 4 ) is one of the few metal-independent restriction endonucleases (REases) found in bacteria. The BmrI restriction-modification system was cloned by the methylase selection method, inverse PCR, and PCR. BmrI REase shows significant amino acid sequence identity to BfiI and a putative endonuclease MspBNCORF3798 from the sequenced Mesorhizobium sp. BNC1 genome. The EDTA-resistant BmrI REase was successfully over-expressed in a pre-modified E. coli strain from pET21a or pBAC-expIQ vectors. The recombinant BmrI REase shows strong promiscuous activity (star activity) in NEB buffers 1, 4, and an EDTA buffer. Star activity was diminished in buffers with 100-150 mM NaCl and 10 mM MgCl 2 . His-tagged BmrI192, the Nterminal cleavage domain of BmrI, was expressed in E. coli and purified from inclusion bodies. The refolded BmrI192 protein possesses non-specific endonuclease activity. BmrI192 variants with a single Ser to Cys substitution (S76C or S90C) and BmrI200 (T200C) with a single Cys at the Cterminal end were also constructed and purified. BmrI200 digests both single-strand (ss) and doublestrand (ds) DNA and the nuclease activity on ss DNA is at least 5-fold higher than that on ds DNA. The Cys-containing BmrI192 and BmrI200 nuclease variants may be useful for coupling to other DNA binding elements such as synthetic zinc fingers, thio-containing locked nucleic acids (LNA) or peptide nucleic acids (PNA).

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BfiI, a restriction endonuclease from Bacillus firmus S8120, which recognizes the novel non-palindromic sequence 5'-ACTGGG(N)5/4-3'

Cited by 18 publications

References 8 publications

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

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

Site-specific DNA transesterification catalyzed by a restriction enzyme

Expression and purification of BmrI restriction endonuclease and its N-terminal cleavage domain variants

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