Novel Subtype of Type IIs Restriction Enzymes

Sapranauskas, Rimantas; Sasnauskas, Giedrius; Lagunavičius, Aru̅nas; Vilkaitis, Giedrius; Lubys, Arvydas; Šikšnys, Virginijus

doi:10.1074/jbc.m003350200

Cited by 94 publications

(59 citation statements)

References 33 publications

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“…Likewise, PTO derivatives of ATP often prevent ATPdependent enzymes from binding Mg 2ϩ (27). BfiI, on the other hand, does not use metal ions in its reactions (12), so factors that affect metal ion binding will not affect its activity.…”

Section: Discussionmentioning

confidence: 99%

“…This REase can therefore not only cleave DNA at specific phosphodiester bonds, but it can also join together two DNA segments by transferring the 5Ј-phosphate at the site of cleavage to a 3Ј-OH from elsewhere in the DNA. The BfiI REase belongs to the PLD superfamily of enzymes (12), and covalent phosphohistidine intermediates have been demonstrated previously for a number of PLD enzymes, including PLD itself, tyrosyl-DNA PDE, and Nuc (18,20,26). Stereochemical pathways for many endonucleases acting at specific DNA sites have been determined by using DNA substrates in which the scissile phosphate is replaced with a chiral phosphorothioate (PTO) of known configuration (27).…”

Section: Dna Alcoholysis By Bfiimentioning

confidence: 99%

“…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.…”

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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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Section: Discussionmentioning

confidence: 99%

Section: Dna Alcoholysis By Bfiimentioning

confidence: 99%

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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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confidence: 99%

“…In contrast, the BfiI restriction endonuclease does not require metal ions: it can cleave DNA in the presence of EDTA (22). 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).…”

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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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Protein NCRII‐18: the role of gene fusion in the molecular evolution of restriction endonucleases

2017

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This work first constructed the fusion protein NCRII-18 by fusing the restriction endonuclease Ecl18kI gene and part of the gene coding for the N-terminal domain of the endonuclease EcoRII. The fusion of the EcoRII N-terminal domain leads to a change in the properties of the recombinant protein. Unlike Ecl18kI, which made the basis of NCRII-18, the fusion protein predominantly recognizes the CCWGG sites, having lost the capability of interacting with the CCSGG sites. Experimental data support the hypothesis of a close evolutionary relationship between type IIE and IIP restriction endonucleases via a recombination between domains with active site structure and elements for recognition with domains responsible for recognition of DNA sequences.

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Novel Subtype of Type IIs Restriction Enzymes

Cited by 94 publications

References 33 publications

Site-specific DNA transesterification catalyzed by a restriction enzyme

Site-specific DNA transesterification catalyzed by a restriction enzyme

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

Protein NCRII‐18: the role of gene fusion in the molecular evolution of restriction endonucleases

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