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Site-Specific DNA Transesterification Catalyzed by a Restriction Enzyme

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Site-Specific DNA Transesterification Catalyzed by a Restriction Enzyme Site-specific DNA transesterification catalyzed by a restriction enzyme Giedrius Sasnauskas*, Bernard A. Connolly†, Stephen E. Halford‡, and Virginijus Siksnys*§ *Institute of Biotechnology, Graiciuno 8, Vilnius, LT-02241, Lithuania; †Institute for Cell and Molecular Biosciences, University of Newcastle, Newcastle upon Tyne NE2 4HH, United Kingdom; and ‡Department of Biochemistry, School of Medical Sciences, University of Bristol, University Walk, Bristol BS8 1TD, United Kingdom Edited by Linda Jen-Jacobson, University of Pittsburgh, Pittsburgh, PA, and accepted by the Editorial Board December 7, 2006 (received for review October 2, 2006) Most restriction endonucleases use Mg2؉ to hydrolyze phosphodi- donor to an acceptor, to either water or an alcohol (19). The ester bonds at specific DNA sites. We show here that BfiI, a phospholipases and the nucleases in this family use water to metal-independent restriction enzyme from the phospholipase D hydrolyze phosphodiester bonds, whereas the phospholipid syn- superfamily, catalyzes both DNA hydrolysis and transesterification thases use an alcohol in a transesterification reaction to make a reactions at its recognition site. In the presence of alcohols such as new phosphodiester. ethanol or glycerol, it attaches the alcohol covalently to the 5؅ PLD proteins are characterized by a conserved sequence motif terminus of the cleaved DNA. Under certain conditions, the termi- called HXK (17, 19), and they use the conserved histidine in the nal 3؅-OH of one DNA strand can attack the target phosphodiester motif for nucleophilic attack on the target phosphorus. They follow bond in the other strand to create a DNA hairpin. Transesterifica- a two-step mechanism (18, 20): in the first step, they attack the tion reactions on DNA with phosphorothioate linkages at the phosphorus to form a covalent phosphohistidine intermediate and target bond proceed with retention of stereoconfiguration at the release the alcohol; in the second, the intermediate is attacked by phosphorus, indicating, uniquely for a restriction enzyme, a two- either another alcohol or water to yield, respectively, the transes- step mechanism. We propose that BfiI first makes a covalent terification or the hydrolysis product (21, 22). enzyme–DNA intermediate, and then it resolves it by a nucleophilic Transesterification reactions have, however, yet to be observed attack of water or an alcohol, to yield hydrolysis or transesterifi- with any PLD enzyme acting on DNA, either tyrosyl-DNA PDE cation products, respectively. (18, 23) or the nucleases (15) in this superfamily. Here we show that alcohols can act as nucleophiles in the DNA cleavage reaction ͉ ͉ ͉ ͉ alcoholysis BfiI covalent intermediate restriction endonuclease catalyzed by BfiI to yield transesterification products. Moreover, we stereochemistry demonstrate that the 3Ј-hydroxyl group of DNA can act as an intramolecular nucleophile to generate a DNA hairpin. Finally, we ϩ n the presence of Mg2 , type II restriction endonucleases show from the stereochemistry of transesterification that, unlike I(REases) hydrolyze specific phosphodiester bonds in DNA to other restriction enzymes, the reaction pathway of BfiI follows a yield 5Ј-phosphate and 3Ј-hydroxyl termini (1). The stereochem- two-step mechanism. BIOCHEMISTRY ical pathway for phosphodiester hydrolysis by four such enzymes, EcoRI, EcoRV, SfiI, and HpaII (2–4), have been determined. Results All proceed with inversion of configuration at the scissile DNA Alcoholysis by BfiI. BfiI has a single active site, which it uses phosphate, which implies an odd number of chemical steps in the sequentially to cut both DNA strands (24, 25). It first cuts the hydrolytic reaction, and it is most simply accounted for by a bottom strand 4 nt downstream from its recognition sequence, Ј direct in-line displacement of the 3 -leaving group by water 5Ј-ACTGGG-3Ј. Then it slowly cuts the top strand at varied (5–7). High-resolution structures are available for many more positions 5–7 nt downstream from the site (25). To study the REases than have been analyzed stereochemically (1). Their reactions of BfiI, a minimal substrate, 14/15 (Table 1 and Fig. 1A), active sites are generally similar, with several carboxylates to act 2ϩ was constructed by annealing 14- and 15-nt oligonucleotides for its as ligands for Mg in spatially equivalent positions (1). All such top and bottom strands, respectively. The duplex contains the enzymes probably act in the same way, by using water to attack recognition site for BfiI, but it can only be cleaved in the bottom the scissile phosphate, to invert its configuration. strand because it lacks the scissile phosphodiester bonds in the top Endonucleases from the transposase-retroviral integrase fam- strand. The 15-nt bottom strand was radiolabeled at its 3Ј end. In ily, like MuA or HIV-integrase, catalyze at a single active site single-turnover reactions in aqueous buffer, BfiI cut this strand at two sets of reactions on DNA (4, 8, 9). The first, the endonu- Ϫ a relatively rapid rate, 2 s 1 (see below and Fig. 4B), to give the 13-nt cleolytic cleavage of the 3Ј end of the transposon or viral DNA, (radiolabeled) product with a 5Ј-phosphate and a dinucleotide with is mechanistically similar to the hydrolysis reactions of REases. a3Ј-OH; no other products were detected (Fig. 1B). 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 Author contributions: G.S. and V.S. designed research; G.S. performed research; B.A.C. DNA (9, 10). contributed new reagents/analytic tools; G.S., S.E.H., and V.S. analyzed data; and G.S., We ask here whether the BfiI REase can catalyze transesteri- B.A.C., S.E.H., and V.S. wrote the paper. fication reactions in addition to DNA hydrolysis. BfiI cleaves The authors declare no conflict of interest. DNA at specified positions downstream from an asymmetric This article is a PNAS direct submission. L.J.-J. is a guest editor invited by the Editorial Board. recognition sequence (11). Unlike other REases, it functions Freely available online through the PNAS open access option. without metal ions (12). The N-terminal half of BfiI (13, 14) is Abbreviations: CIAP, calf intestine alkaline phosphatase; ddA, dideoxyadenosine; PDE, similar to Nuc, an EDTA-resistant nuclease from Salmonella phosphodiesterase; PLD, phospholipase D; PNK, polynucleotide kinase; PTO, phosphoro- typhimurium (15, 16) that belongs to the phospholipase D (PLD) thioate; REase, restriction endonuclease. superfamily. The PLD superfamily is a diverse group of proteins §To whom correspondence should be addressed. E-mail: [email protected]. that includes phospholipases, phospholipid synthases, bacterial This article contains supporting information online at www.pnas.org/cgi/content/full/ toxins, phosphodiesterases (PDEs), and nucleases (17, 18). The 0608689104/DC1. PLD enzymes catalyze phosphoryl transfer reactions from a © 2007 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0608689104 PNAS ͉ February 13, 2007 ͉ vol. 104 ͉ no. 7 ͉ 2115–2120 Downloaded by guest on September 26, 2021 Table 1. Oligonucleotide duplexes and yields of hairpin DNA products in BfiI reactions Max. hairpin Duplex Oligonucleotide duplex sequence* yield, % 14/15 5Ј-AGCACTGGGCTGC–T-3Ј 0 3Ј-TCGTGACCCGACGpAC-5Ј 25C/15 5Ј-AGCACTGGGCTGC–TGAACTAGTTCA-3Ј 38 Ϯ 2 3Ј-TCGTGACCCGACGpAC-5Ј 25CddA/15† 5Ј-AGCACTGGGCTGC–TGAACTAGTTCddA-3Ј 0 3Ј-TCGTGACCCGACGpAC-5Ј 15/15 5Ј-AGCACTGGGCTGC–TG-3Ј 0 3Ј-TCGTGACCCGACGpAC-5Ј 16/15 5Ј-AGCACTGGGCTGC–TGA-3Ј 5.5 Ϯ 1 3Ј-TCGTGACCCGACGpAC-5Ј 17/15 5Ј-AGCACTGGGCTGC–TGAG-3Ј 14 Ϯ 1 3Ј-TCGTGACCCGACGpAC-5Ј 25/15 5Ј-AGCACTGGGCTGC–TGAACTGTGCTG-3Ј 28 Ϯ 3 3Ј-TCGTGACCCGACGpAC-5Ј 29/15 5Ј-AGCACTGGGCTGC–TGAACTGTGCTGCGGC-3Ј 1.4 Ϯ 0.5 3Ј-TCGTGACCCGACGpAC-5Ј 25C/15(S)Rp‡ 5Ј-AGCACTGGGCTGC–TGAACTAGTTCA-3Ј 36 Ϯ 5 3Ј-TCGTGACCCGACGsAC-5Ј 25C/15(S)Sp§ 5Ј-AGCACTGGGCTGC-TGAACTAGTTCA-3Ј 37 Ϯ 5 3Ј-TCGTGACCCGACGsAC-5Ј *The BfiI recognition sequence is underlined, and the scissile phosphodiester bond in the bottom DNA strand is noted as p. Unless noted otherwise, all duplexes carried a radiolabel at the 3Ј terminus of the bottom strand. †The 3Ј-terminal nucleoside of the top strand of 25CddA/15 is dideoxyadenosine (ddA). ‡ The 25C/15(S)Rp duplex contains an Rp–PTO linkage at the scissile position of the bottom strand (marked s). § The 25C/15(S)Sp duplex contains an Sp–PTO linkage at the scissile position of the bottom strand (marked s). When BfiI reactions on 14/15 were conducted in the presence of bond, which may in turn be cleaved by BfiI. When challenged various alcohols, additional products were observed by gel electro- with BfiI, the additional products were cleaved to the normal phoresis, with mobilities between those of the 15-nt substrate and product with a 5Ј-phosphate, albeit at rates that were Ն40-fold the 13-nt product (Fig. 1B). The mobilities of the extra bands varied slower than the native phosphodiester in DNA (see SI Fig. 8). inversely with the molecular mass of the alcohol: Tris gave the slowest extra band and ethanol the fastest, with glycerol and 1,2-ethanediol at intermediate values. In all cases, the yield of extra product increased proportionally with the alcohol concentration (Fig. 1B). The additional species generated during DNA cleavage in the presence of alcohols may therefore be transesterification products formed by the transfer of the scissile phosphate to the alcohol, rather than to water, to leave the alcohol attached to the 5Ј-phosphate of the 13-nt product. This reaction was confirmed by mass spectrometry, which revealed two 13-nt products from the reaction with each alcohol: one with the same mass as the hydrolysis product and one with an increased mass. In each case, the increase in mass matched the alcohol in question [see supporting informa- tion (SI) Fig.
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