1New England Biolabs, Inc. 240 County Road, Ipswich, MA 01938, USA and 2Department of Microbiology and Molecular Medicine, Center of Excellence in Bacteriology of the University of Geneva, University of Geneva, Faculty of Medicine, 1, rue Michel Servet 1211 Geneve 4, Switzerland. The digestion was incomplete because many of the bands are bigger than the predicted cleavage products of 5mCCGG, C5mCGG, G5mCNGC, C5mCNGG or C5mCWGG generated by virtual digestion using the NEB cutter (http://tools.neb.com/NEBcutter2/). Protein gel analysis of SauUSI variants and activity assay. Supplementary Data are available at NAR Online. Abstract. http://creativecommons.org/licenses/by-nc/2.5, supp_gkr098_SauUSI_supplement_figures_1_to_7.pdf, R gene inserted in opposite orientation to Tc. In general, restriction enzymes cleave double-stranded DNA. 1. They are required in genetic mapping and reconstruction of the DNA in vitro only because they identify particular sites and cleave at those sites only. A type III-like restriction endonuclease functions as a major barrier to horizontal gene transfer in clinical, Bidnenko E, Chopin A, Ehrlich SD, Chopin MC. This open reading frame (ORF) contains a super family II DNA helicase domain and was found to be responsible for biological restriction of plasmid transformation when DNA was prepared from E. coli sources and suggested to be a type III restriction endonuclease based on the presence of helicase superfamily II domain. We also tested SauUSI digestion of plasmids that have been modified in vivo by C5 methylases. 2. Liu G, Ou H-Y, Wang T, Li L, Tan H, Zhou X, Rajakumar K, Deng Z, He X. Cleavage of phosphorothioated DNA and methylated DNA by the type IV restriction endonuclease ScoMcrA. (C) SauUSI digestion of in vitro modified DNA. Lanes 12 and 23, 2-log DNA size ladder. (B) T4gt DNA was digested by SauUSI at various ATP concentrations (0.220mM). Some PLD family nucleases are active in the absence of divalent metal ions (in the presence of EDTA) (31,32). Now, these copies can be utilized for further analysis of whatsoever type. National Library of Medicine Staphylococcus aureus ST398 strain carries a putative RM system with the predicated target sequence of CCNGG (the putative C5 methylase displays 69% aa sequence identity to M1.ScrFI, CCNGG). (A) SDSPAGE gel analysis of batch purified SauUSI catalytic mutants H119A, K121A, N139A, E150A,and the wt enzyme (two isolates for each mutant are shown). Inspection of the sequenced genome of S. aureus subsp. Dcm+ vir (a gift from A. Fomenkov, Mern Sibley, Lise Raleigh) was prepared from E. coli host ER1793. No data on intracellular ATP concentration were available for S. aureus USA300 strain. . Type IIB restriction enzymes (e.g., BcgI and BplI) are multimers, containing more than one subunit. Within the Type II category, there are up to 8 different subdivisions that have slightly different recognition site and cleavage properties. Hinf III, EcoP1. Tel: +1 978 380 7287; Fax: +1 978 921 1350; Email: Received 2010 Dec 2; Revised 2011 Feb 3; Accepted 2011 Feb 4. Examples Hinfl, EcoRI, PvuII, Alul, Haelll. This experiment has some biological implication in the transfer SauUSI-like restriction genes among bacteria. This group is an intermediate type between type I and type II. However, to break the double-stranded DNA, two recognition sites in opposite directions are required. The results suggest that as long as SauUSI expression level is tightly controlled, sauUSIR-like genes could be acquired by 5mC-methylase carrying hosts. Modified pBC4 DNAs by M.SssI (5mCG) or M.CviPI (G5mC) also allowed partial cleavage by SauUSI (data not shown). Two separate enzymes mediate restriction and modification. And what makes them so useful? The target sequence is then detected by a combination of two processes. Cleavage patterns of HindIII, SmaI, EcoRI, and BamHI are described as below. They require both AdoMet and Mg 2+ cofactors. REBASE (Restriction Enzyme dataBASE: http://www.neb.com/rebase )-assisted restriction mapping is described in this paper for a rare cutter [TspMI (REBASE No. Each restriction enzyme recognizes a specific sequence of 4-8 nucleotides in DNA and cleaves at these sites. Therefore, incomplete modification does not explain resistance to SauUSI cleavage. The recognition and modification of DNA are carried out by the first subunit- M and the nuclease activity is rendered by the other subunit R. Although the cleavage appears to be non-specific, the initial incision (nicks or ds breaks) displays certain sequence preferences, with S5mCNGS as the preferred sites. The relative endonuclease activity in different metal ions are Mg++>Mn++ > Ca++>Co++ (data not shown). Table 4 shows the transformation results. Recognition sequence 5-GAATTC-3 3-CTTAAG-5 ; sticky ends, Recognition sequence 5-CCWGG-3 3-GGWCC-5; sticky ends, Recognition sequence 5-GGATCC-3 3-CCTAGG-5: sticky ends, Recognition sequence- 5 TCGA-3 3-AGCT-5, Recognition sequence 5 AAGCTT-3 3-TTCGAA -5. Fig: Restriction Enzyme These enzymes have the capacity to recognize the specific base sequences on the DNA helix and then cut each strand at a given place. In addition, we found that divalent cations are required for optimal endonuclease activity. Lanes 5, 6, 811, 1317, 19, modified plasmid mixture (plasmid with M gene plus pBC4) digested by SauUSI. Digestion of T4gt by SauUSI in NEB buffers 14, in the presence of EDTA, or in the absence of metal ions (depleted from enzyme preparation). The nucleotides are cleaved at the restriction site only. It consists of two homodimers each 30kDa molecular mass, that recognize the palindromic sequences. To exclude problems in transformation efficiency other than the restriction of the plasmid DNA, we also prepared plasmid DNA from RN4220, the permissive laboratory S. aureus strain. Abstract This article continues the series of Surveys and Summaries on restriction endonucleases (REases) begun this year in Nucleic Acids Research. These are usually synthesized by bacteria for defense mechanisms against invading bacteriophages. A Superdex 200 5/150 GL (3ml bed volume; GE Healthcare) was run with NEBuffer 4 at 4C at a flow of 0.2ml/min. Lane 7, M.BbvI-modified plasmid pUC-bbvIM; lane 18, M.TspRI-modified plasmid pUC-tspRIM (M gene under native Thermus promoter). PCR substrates were digested by SauUSI for at 37C for 2h and the enzyme was inactivated at 65C for 20min. 4. Lanes 9 and 10, T4gt digested with two concentration of batch purified wt enzyme; lane 11, wt SauUSI (column purified). The two parts are separated by a non-specific spacer of about 6-8 nucleotides. The results presented in Table 3 show that the transformation efficiency of Dcm+-modified DNA is reduced by more than three-order of magnitude in wt SA564 strain. Type II Restriction Enzymes. Lanes 13, unmodified DNA digested by SauUSI. They can also used for gene therapy, to cut and paste DNA sequences into a new vector. 2) Type II restriction enzymes. The most convenient restriction endonuclease enzymes belong to the type II enzyme. SauUSI does not cleave N4mC and N6mA-modified DNA (the Dcm- substrates do carry adenine modification in the G6mATC context). They cleave DNA on both sides of their recognition to cut out the recognition site. The name contains the genus, species, and the strain of the bacterium. The column was run with NEB buffer 4 at 4C at a flow rate of 0.2ml/min. SauUSI displays a strong nuclease activity on single-stranded DNA. Lagunavicius A, Sasnauskas G, Halford SE, Siksnys V. The metal-independent type IIs restriction enzyme BfiI is a dimer that binds two DNA sites but has only one catalytic centre. to indicate the serotype or strain, a space, then a Roman numeral to indicate the chronology of identification. If you would like to change your settings or withdraw consent at any time, the link to do so is in our privacy policy accessible from our home page.. Type IIP enzymes specific for 6-8 bp sequences mainly act as homodimers, composed of two identical protein chains that associate with each other in opposite orientations (Examples: EcoRI, HindIII, BamHI, NotI, PacI.) If so, activation might allow action in trans on a separate unmodified molecule. Henceforth, DNA can be cleaved in the absence of modifying enzymes. Based on the composition, characteristics of the cleavage site, and the cofactor requirements, the restriction endonucleases are classified into four groups, Type I, II, III, and IV. For its activity some requires magnesium ions while others required ATP and S-adenosyl L- methionine. Such mutants would be useful for epigenetic studies. The specific activity of SauUSI is determined to be 8000 U/mg protein on T4gt phage DNA in the presence of ATP. and transmitted securely. Lowry OH, Carter J, Ward JB, Glaser L. The effect of carbon and nitrogen sources on the level of metabolic intermediates in. Figure 3A shows that only ATP or dATP supports SauUSI endonuclease activity. It is possible to select SauUSI mutants that prefer to cleave 5hmC DNA. These ends are often ligated by DNA ligase enzymes. This results in a fusion of the ORF to the Sce VMA intein and Bacillus circulans CBD (IMPACT protein purification system, NEB). The specific palindromic site can either be continuous (e.g., KpnI identifies the sequence 5-GGTACC-3) or non-continuous (e.g., BstEII recognizes the sequence 5-GGTNACC-3, where N can be any nucleotide). Roberts RJ, Vincze T, Posfai J, Macelis D. REBASE a database for DNA restriction and modification: enzymes, genes and genomes. Before N4mC modified DNA by M.BamHI (GGATm4CC) or N6mA modified by M.TaqI (TCG6mA) are poor substrates for SauUSI (Figure 1C, lanes 4 and 7). For the M.Fnu4HI-modified plasmid DNA (6kb), the SauUSI cleavage sites vary from N3 to N18 (G5mCNGC N318, bottom strand). SauUSI is a multi-domain protein with a PLD-family endonuclease catalytic site located at the N-terminal domain, a DNA helicase/ATPase located in the middle part of the protein, and a presumed 5mC/hmC TRD domain (DUF3427) located at the C-terminus. Restriction enzymes are important for a variety of reasons. A gene encoding a putative DNA helicase from Staphylococcus aureus USA300 was cloned and expressed in Escherichia coli. To study the cofactor requirement of SauUSI endonuclease activity, EDTA-treated and extensively dialyzed SauUSI enzyme was incubated with DNA substrates in reaction buffers containing different divalent cations (1 and 5mM of Mg++, Mn++, Ca++, Co++, Zn++ or Ni++). What are Restriction Enzymes? Abstract. We report the characterization and cloning of the genes for an unusual type IV restriction-modification system, BspLU11III, from Bacillussp. More distant SauUSI orthologs can be found in over 150 sequenced bacterial/archaea genomes. Restriction Enzyme Resource This guide introduces restriction enzymes, providing i n-depth reference information and tools to help you find buffers for double digests, or find enzymes by name or recognition sequence. A novel family of phospholipase D homologues that includes phospholipid synthases and putative endonucleases: identification of duplicated repeats and potential active site residues. The protein was diluted in NEB Diluent A buffer and stored at 20C. Examples EcoK, EcoB. 5mC- or 5hmC-modified PCR DNAs with S5mCNGS sites (1, 2, 4, 5 and 6 sites) were produced in PCRs with 5mC (5m-dCTP) or 5hmC (5hm-dCTP) instead of dCTP. Due to its nucleolytic activity these enzymes are extensively used in recombinant DNA technology. These regions are called recognition sequences, or recognition sites, and are randomly distributed throughout the DNA. Type I and Type III restriction/modification enzyme systems are less known and less common, but biologically very curious. Plasmid pBR-sauUSIR** carries a single base deletion at nt position 85, causing a frame-shift mutation in the SauUSI endonuclease. Each restriction enzyme recognizes a short, specific sequence of nucleotide bases (the four basic chemical subunits of the linear double-stranded DNA molecule adenine, cytosine, thymine, and guanine ). More cleavage sites remain to be analyzed in order to get a complete picture of SauUSI cut sites. Inclusion in an NLM database does not imply endorsement of, or agreement with, As restriction enzymes are target-specific, 15-20 hydrogen bonds are formed between the dimers and bases of the target site. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (, GUID:158DE0AA-C50F-4A02-A030-1F00C9D024A0, GUID:4D75159B-AD35-48A1-9D28-40ED941F3237, GUID:2FED13BD-F7A5-42E6-895F-8AC84FAC892B. This 5mC/hmC-dependent substrate specificity is shared by McrBC, although McrBC activity requires GTP hydrolysis. For small-scale affinity purification, cell extracts from 10ml IPTG-induced overnight culture (16C) were used for batch purification of the mutant enzymes. Raleigh EA. A relative molecular weight of 76kDa was obtained from the standard curve (the insert equation). DNA fragments were resolved in 2.5% agarose gel electrophoresis. The purified enzyme, SauUSI, predominantly cleaves modified DNA containing 5mC and 5-hydroxymethylcytosine. This type of restriction enzyme are multifunctional proteins that cleave only one DNA strand at random as well as distant sites and also performs methylase activities. They are composed of two different subunits. Lane 7, SauUSI digestion in the NTD buffer (50mM NaCl, 20mM TrisHCl, pH 8.0, 1mM DTT), plus 10mM EDTA. We are grateful to Lise Raleigh, Mern Sibley, Alexey Fomenkov, Rick Morgan, Keith Lunnen, Geoff Wilson, Lucia Greenough, Bill Jack for strains and plasmids. However, amino acid sequencing has uncovered extraordinary variety among restriction enzymes and revealed that at the molecular level, there are many more than four different types. Genomic DNA of S. aureus subsp. The purification results are shown in Supplementary Figure S1. (D) SauUSI digestion of dC-, 5mC-, 5hmC-containing PCR DNA (207bp-4 sites). The flow-through containing the SauUSI protein was then loaded onto a HiTrapTM heparin column (GE Healthcare), and proteins were eluted with a linear gradient of 50mM to 1M NaCl. We tested the restriction of phages by SauUSI endonuclease using Dcm+ or Dcm phages that have been grown on modification proficient or deficient E. coli hosts. The type II restriction enzymes first establish non-specific contact with DNA and bind to them in the form of dimmers. Supplementary Figure S6 (top panel) shows the DNA sequencing result from uncut pBR322 DNA. These enzymes are the most stable endonucleases which cleave DNA at specific sites. Over 150 proteins with 25100% amino acid sequence identity with a similar functional domain organization are found in sequenced bacterial genomes. We also tested a number of N4mC-modified plasmids in SauUSI digestion. The flow-through was then loaded onto a HiTrapTM heparin column and protein eluted with a NaCl gradient. Type II REases with 48-bp recognition sequences and over 300 unique specificities are widely used in creating recombinant DNA molecules (4). Methylases involved with these processes (for example Dam and Dcm methylases) are independent from the restriction modification systems, yet can still affect whether certain restriction enzymes can effectively cleave DNA. Less sensitive to SauUSI cleavage are M.AclI (AA5mCGTT), M.HpyCH4IV (A5mCGT) and M.NspHI (R5mCATGY). Further experiments are needed to uncouple the two reactions. Once the target sequence is located, various conformational changes occur in the enzyme as well as the DNA. The and + on top of pUC indicate unmodified or modified pUC19 by Dcm methylase, respectively. However, this nuclease activity is not modification dependent (data not shown). We mutated H119, K121, N139 and E150 to Ala by site-directed mutagenesis and partially purified the mutant proteins. The digestion pattern, appearing as a smeargram, suggests non-specific nuclease activity (or strong star activity) following incision from (or near) the modified 5mC/5hmC sites. Bethesda, MD 20894, Web Policies For small-scale protein purification, 4L cells of ER2566 [pTYB1-SauUSIR] were cultured at 37C in LB supplemented with Amp (100g/ml) until late log phase. New England Biolabs, Inc. Since SauUSI contains a helicase domain and ATP-binding site (inferred from amino acid sequence similarity to other type I and III REases), we included ATP (1mM) in all digestions (see below for further data on nucleotide requirement). Lane 9, 2-log DNA size marker. Figure 2B shows undigested plasmid DNAs. The recognition sequences are generally 4 to 8 base pairs (bp) in length, and cleavage can produce sticky ends (5 or 3 . Dcm SauUSI is a 5mC/5hmC modification-dependent REase. A Roman numeral to indicate the order in which the different restriction-modification systems were found in the same organism or strain per se. T4gt deficient in - and -glucosyl transfereases contains 5hmC DNA. Dcm+Dam, Dcm+Dam+ and pBR322 Dcm+ DNAs were from NEB. Transformation efficiency of pBR-sauUSIR, pBR-sauUSIR* and pBR322 plasmids into Dcm+ E. coli strain NEB10. Some enzymes cut DNA at their recognition site, while others have separate recognition and cleavage sites. Liu G, Ou HY, Wang T, Li L, Tan H, Zhou X, Rajakumar K, Deng Z, He X. Cleavage of phosphorothioated DNA and methylated DNA by the type IV restriction endonuclease ScoMcrA. SauUSI digestions of various DNA substrates were carried out at 37C in NEB buffer 4, except specified otherwise. Database resources of the National Center for Biotechnology Information. Those that are sensitive to cleavage are M.BssKI (C5mCNGG or 5mCCNGG), M.TseI (G5mCWGC) and M.BsrFI (RC5mCGGY) (Figure 2A). IPTG-induced ER2566 cell extracts showed endonuclease activity on DNA (Dcm+) in the presence of ATP and 10mM DTT (data not shown). Figure 1C shows that SauUSI cleaves pBC4 plasmid after in vitro modification by M.MspI (5mCCGG), M.HpaII (C5mCGG), M.HhaI (G5mCGC) or M.AluI (AG5mCT). Two heparin fractions with the highest purity (Supplementary Figure S1, lanes 5 and 6) were concentrated to 0.2mg/ml and used for further characterization. SauUSI is active in the four NEB buffers, all of which contain 10mM Mg++ (before EDTA treatment and dialysis), with highest activity in buffer 4 (50mM potassium acetate). The 0.40.5-kb DNA of T4gt digested products was an artifact which dispersed in high percentage agarose gel or PAGE (data not shown). The metal ion-depleted SauUSI regained activity in buffer 4 with Mg++, and its activity was again inhibited if EDTA was added (Figure 4A, lanes 10 and 11). The recognition sequences are palindromic in nature of 4-8 bp and for catalytic activity, only Mg2+ ions are required. Restriction and modification systems. This creates DNA fragments with one nucleotide strand that overhangs at the end. The melt temperature defines the point at which half of the DNA is single-stranded. After centrifugation, clarified cell lysate was loaded onto a chitin column (21ml, 4cm 2.6cm), which was then extensively washed with 10 column volumes of column buffer (20mM TrisHCl, pH 8.5, 0.5M NaCl). Each protein subunit binds roughly one-half of the recognition sequence and cleaves one DNA strand. For example, Hind III was the third of four enzymes isolated from Haemophilus influenza serotype d. 6. With 41.5 C being the low range, we prefer that the enzyme be able to work at room temperature. This was demonstrated with a mixing experiment. Expression and purification of BmrI restriction endonuclease and its N-terminal cleavage domain variants. A restriction enzyme also known as molecular scissors is a site-specific endonuclease encoded by bacteria and archaea. 1b). SauUSI-digested PCR DNAs were purified by spin columns using a PCR purification kit (Qiagen) and run-off sequencing was carried out using a Big-Dye terminator cycle sequencing kit (ABI/Life technologies). Restriction Enzyme Tools are available for desktop or mobile. For batch purification of wt SauUSI and variants using chitin beads, 10ml of cells were cultured to late log phase at 37C. To remove the nucleic acids from the eluted proteins, the pooled fractions were loaded onto a Source Q column (GE Healthcare) at 0.25M NaCl concentration. T4gt phage containing 5hmC was prepared from ER2566. EcoRI . Sau1: a novel lineage-specific type I restriction-modification system that blocks horizontal gene transfer into, Corvaglia AR, Francois P, Hernandez D, Perron K, Linder P, Schrenzel J. The cleaved and eluted fractions were passed through a Source Q column at moderate salt concentration to remove most of the nucleic acids. Lane 2, pUC-bbvIM; lane 7, pBR-fnu4HIM. The absence of a cognate methylase and cleavage of modified DNA indicate that SauUSI belongs to type IV restriction endonucleases, a group that includes EcoK McrBC and Mrr. Genetic analysis of a high-level vancomycin-resistant isolate of. The biological relevance of the preference of ATP/dATP over GTP is not clear. The recognition sites are typically a short palindromic sequence of 4-8 bp and catalytic activity requires Mg2+ ions. Lane 10, T4gt DNA digested with metal ion-depleted SauUSI in buffer 4. 5hmC-containing PCR DNA with two sites was also cleaved by SauUSI (data not shown). Phage restriction activity of SauUSI endonuclease. It might be advantageous not competing for the same cofactor if SauUSI-like and McrBC-like type IV restriction systems co-exist in the same bacterium. Lane 12, uncut T4gt DNA substrate. The first two letters of the species name are written after the first initial. Desired mutations were confirmed by DNA sequencing. document.getElementById( "ak_js_1" ).setAttribute( "value", ( new Date() ).getTime() ); DNA Replication Enzymes of Prokaryotes and Their Roles, Escherichia coli (E. coli) Types and Uses in Biotechnology, Difference Between a Cloning Vector and an, List of School Science Laboratory Equipment and. Restriction enzymes are utilized for gene insertion into plasmids during cloning and protein expression experiments. Activation of mRNA translation by phage protein and low temperature: the case of. Restriction enzyme, which is also known as Restriction Endonuclease, is a protein that is produced by bacteria that cleaves DNA at specific sites along the molecule. Lanes 1, 36, 812, plasmid with the indicated M gene and pBC4 mixture. Inactivation of this gene by mutation increased the transformation efficiency by 102104-fold. We measured the transformation efficiencies of plasmid prepared from different E. coli strains. Proteins encoded by the DpnI restriction gene cassette. The endonuclease catalytic activity is tightly coupled with 5mC or 5hmC recognition. Zheng Y, Cohen-Karni D, Xu D, Chin HG, Wilson G, Pradhan S, Roberts RJ. This group comprises most orthodox restriction enzymes which are used in recombinant DNA technology. The resultants of the catalysis are DNA fragments with 5-P and 3-OH. NEB currently offers over 50 Type IIS restriction enzymes. Similarly, after digestion of pBR-fnu4HIM (G5mCNGC) plasmid by SauUSI endonuclease, the digested DNA was purified by spin column and used as template for run-off sequencing to determine the cleavage sites. 8600 Rockville Pike Type I REs are important in bacterial function but do not cleave DNA at specific sequences. Efficient cleavage requires more than one site; and the cleavage sites outside of its recognition sequence appear to be variable (N2N18). Supplementary Figure S6 (bottom panel) shows that SauUSI cleaves outside of recognition sequence at N3, N10 and N1418, with complete run-off at N18 from the target site, indicating SauUSIs ability to travel and cleave at long distance from the G5mCCGC site. Restriction enzyme is a site- specific endonuclease that cleaves DNA strands at specific recognition sites. Phages were diluted in phage broth or 10mM MgCl2 solution and used to infect the E. coli hosts. These are synthesized by bacteria as a part of their defense mechanism. A large number of studies have been carried out to elucidate the mechanism of antibiotic resistance. Mixtures of plasmids prepared from E. coli and S. aureus were transformed into the clinical strain SA564 and its restriction-deficient counterpart (SA564 hsdR::targetron/type I-deficient, type IV-like::targetron/deficient in SauUSI-like REase) (24). They are large multimeric proteins capable of cleaving and modifying DNA. Eg., McrBC. (C) SauUSI digestion of dC-, 5mC-, 5hmC-containing PCR DNA (73bp-1 site): 100bp, 100bp DNA ladder. (A) Plasmids prepared from co-transformants of pACYC-M (or pSYX20-M) and pBC4 were digested by SauUSI for 1h at 37C and the digested DNAs were analyzed on a 1% agarose gel. The recognition sequence is rotationally symmetrical, called palindromic sequence. The mutant protein yields are 45-fold higher than the wt enzyme, probably reflecting the reduced toxicity of the mutant endonucleases toward the E. coli expression host. One part of the recognition site is composed of 3-4 nucleotides while the other one contains 4-5 nucleotides. It is followed by a letter, number or combination of both of them to signify the strain of the species. There are two different kinds of restriction enzymes: The first restriction enzyme to be discovered was Hind II in the year 1970. To characterize the presumed nucleotide requirement, we tested SauUSI activity on T4gt DNA using different NTP or dNTP in the digestion. Dcm+ pBR322 or pBC4 additionally modified in vitro by M.MspI (5mCCGG) serves as a better substrate for SauUSI digestion than Dcm+ modified alone (data not shown). 7191)] and a frequent cutter [BflI (REBASE No. Eg. SauUSI displays lower cleavage activity on 5-glc-hmC DNA (glucosylated T4 DNA, wild type) (Figure 1A, lane 1).Unmodified (Dcm) pBC4 is a poor substrate for SauUSI although a small fraction of DNA was partially linearized (Figure 1A, lane 5), which suggests that SauUSI has a non-specific endonuclease activity at high enzyme concentration, or that there is a low level of contaminating non-specific nuclease. To view the purposes they believe they have legitimate interest for, or to object to this data processing use the vendor list link below. . Type I restriction enzymes possess both restriction and modification activities. de la Campa AG, Springhorn SS, Kale P, Lacks SA. Duplicated DNA samples: lane 5, pACYC-aclIM; lane 6, pSYX20-aclIM; lane 17, pACYC-tspRIM; lane 18, pUC-tspRIM (M gene under Thermus sp. vir (a gift from A. Fomenkov, Mern Sibley, Lise Raleigh) was prepared from WGG (W = A or T). We used strains deficient in adenine methylation (dam), deficient in cytosine methylation (dcm), a double mutant (dam dcm), or wild-type (wt, dam+ dcm+ DH5) for both methylation systems. The partition coefficient (Kav) of the standard proteins ovalbumin (43kDa), conalbumin (75kDa) and ferritin (440kDa) obtained from two independent runs is plotted against log(mw) (filled diamonds). However, in bacteria, restriction enzymes are present as a part of a combined system called the restriction modification system. Plasmid pBC4 was methylated in vitro by the indicated methyltransferase and subsequently digested by SauUSI. The recognition sequence of SauUSI is therefore somewhat similar to BisI (G5mC/NGC) and BlsI (G5mCN/GC) (9). Digestion of 5mC-modified plasmid DNAs by SauUSI. (24). Additionally, restriction enzymes can used for genetic engineering, to create new recombinant DNA molecules. Polymerase Chain Reaction: 9 Important Explanations, Different Types of PCR: Important Conceptual MCQs. A small-scale purification procedure was developed. The bacterial species modify their own DNA with the help of enzymes which methylate it. For full treatment, see protein: Enzymes. The target sequence is about 15 bp in length and cleaves non-specifically away from the recognition site. The phosphodiester bond is hydrolyzed, and the product is released. In contrast, plasmid DNA prepared from dcm-deficient strains efficiently transformed wild-type and mutant SA564 strains. Another clone with the sauUSIR gene inserted in the opposite orientation was also constructed. From the noncharacterized genes of the Hib capsule gene cluster (bcs2-4), only bcs3 codes for an enzyme large enough (1,215 amino acids) to encompass at least two catalytic activities (Fig. *To whom correspondence should be addressed. The reactions were analyzed using a 5% TopVision gel (Fermentas) stained with SYBR Gold (Invitrogen). Common lab E. coli K12 strains such as DH5alpha contain 3 methylases that recognize and methylate different stretches of DNA: The sequences of recognition sites are palindrome sequences which reads the same on forward and reverse strands when read in the same orientation. In 1978, Daniel Nathans, Werner Arber, and Hamilton O. Smith were awarded the Nobel Prize for Physiology or Medicine. A standard curve was created by obtaining the Kav value of the standard proteins ovalbumin (43kDa), conalbumin (75kDa) and ferritin (440kDa) (GE Healthcare) run under the same condition. LU11. Types of Restriction Enzymes. The general conclusion is that drug resistance genes can be chromosomally encoded or derived from extra chromosomal elements, and acquired by horizontal gene transfer of mobile genetic elements such as conjugative plasmids, transposable elements, integrons or by phage infection and integration (22). Dam+ Dcm pUC19 DNA was poorly digested when it was mixed with DNA (Dcm+) or T4gt (5hmC) DNA, while DNA (Dcm+) or T4gt (5hmC) DNA was preferentially cleaved by SauUSI in the two-DNA mixtures (Figure 4B, lanes 14). Plasmid pBR-sauUSIR** carries a single base deletion at nt position 85, causing a frame-shift mutation in the SauUSI endonuclease (the mutation was introduced in PCR). Finally, we demonstrated the biological function of the type IV REase in restricting 5mC-modified plasmid DNA by transformation into clinical S. aureus strain SA564, and in restricting phage infection when the endonuclease is expressed in E. coli. In contrast, the PCR product made using regular dCTP was only much less cleaved at the same enzyme concentrations, confirming the preference of SauUSI to 5mC-modified DNA (Figure 5A). A single site (GCCGC) with 5hmC incorporated in PCR was a poor substrate for SauUSI digestion (Figure 5C), whereas 5hmC-containing PCR DNA with four SauUSI sites were cleavable by SauUSI (Figure 5D). Lane 11, same as lane 10, but supplemented with 10mM EDTA. These are frequently used for cloning purposes in biotechnology. Examples of type IV are McrBC, which cleaves 5mC-modified DNA, and GmrSD that attacks glucosylated-hmC (glc-5hmC) T4 DNA (1115). Mutations of these four catalytic residues to Ala abolished the endonuclease activity. Plasmid pXbaI was purified from a Dcm+ strain (5mC-modified control DNA). Generally all restriction enzymes are prefixed with the general symbol R. This is used to distinguish from the methylases that are obtained from the same strains. We are excited to announce that all reaction buffers are now BSA-free. They are classified into five types based on their structure and their mode of action. There are three possibilities: (i) interference with gene transfer operates by a mechanism other than cleavage [abortive infection, for example, in references (25,26)]; (ii) interference may be due to site-specific cleavage of an unmodified site, with the M gene located somewhere else on the bacterial chromosome; or (iii) cleavage may occur but depend on specific DNA modifications such as 5mC, 5hmC, glc-hmC, N6mA, N4mC or phosphothioation of the DNA backbone (27). DNA strand contains two strands. The number of enzymes produced by the bacterium. With the advancement in biotechnology, restriction enzyme has become an indispensable tool for recombinant DNA technology. Cell pellets were resuspended in 1ml of cell lysis buffer (20mM TrisHCl, pH 8.5, 0.5M NaCl, 0.1% Triton X-100), and cells were lysed by sonication. The presence of type I, and II RM systems or type IV restriction systems in clinical S. aureus strains could provide a genetic barrier to the free genetic exchange by transformation, transduction and conjugation (23,24). isopropyl--d-thiogalactopyranoside (IPTG) induction was carried out at 0.5mM final concentration and cell cultures induced at 16C overnight. In principle, SauUSI could have a cryptic endonuclease activity that is activated upon recognition of modified DNA. "Restriction enzymes are the enzymes produced by certain bacteria that have the property of cleaving DNA molecule at or near specific base sequences." Read on to explore what are restriction enzymes, their types and applications. Sayers EW, Barrett T, Benson DA, Bryant SH, Canese K, Chetvernin V, Church DM, DiCuccio M, Edgar R, Federhen S, et al. The mechanism comprising methylation along with restriction enzyme activity constitutes the restriction-modification system. Similar results were obtained with 5hmC-containing PCR DNAs. This domain can be found in 255 proteins in the DUF3427 cluster (www.kegg.org) among bacteria and archaea (see Supplementary Table S1 for a list of SauUSI homologs found in many pathogenic G+ bacterial strains). The SauUSIR gene (2859bp, 953aa) flanked by SalI and XhoI restriction sites was amplified by polymerase chain reaction (PCR) using high-fidelity Phusion DNA polymerase (Finnzymes). LM, low molecular DNA marker; 10bp, 10bp DNA ladder. (A) Lanes 14, SauUSI digestion in NEB buffers 1 to 4. The ligated DNA was transferred into ER2566 competent cells by transformation. We focus on their biochemistry: what they are, what they do, and how they do it. We and our partners use cookies to Store and/or access information on a device. Modification-dependent REases are presently grouped into type IIM if the target sequence recognition and cleavage are very specific and precise, for example, DpnI, GN6mATC (8) and GlaI, G5mCG5mC (9), MspJI, 5mCNNR (10); or type IV if cleavage is non-specific and variable. (A) DNA substrates digested by SauUSI endonuclease in 1X NEB buffer 4 at 37C for 1h. (B) Uncleaved DNA substrates. Required fields are marked *, This type of restriction enzyme are multifunctional proteins that cleave only one DNA strand. Ponting CP, Kerr ID. Cleavage takes place nearly 1000 base pairs away from the restriction site. The non-specific nuclease activity might be important to completely destroy the foreign invading DNA (the cleavage products are not easily repaired by simple religation). After analysis of the eluted fractions on SDSPAGE, the fractions with SauUSI were concentrated in a protein concentrator (Amicon/Millipore, 10000kDa) by low-speed centrifugation. Dam modification does not inhibit cleavage since both Dam+ and Dam DNA were equally cleavable by SauUSI (Figure 1A, lanes 3 and 4). Type II enzymes are the most abundant and well-characterized of all restriction enzyme categories. Humbelin M, Suri B, Rao DN, Hornby DP, Eberle H, Pripfl T, Kenel S, Bickle TA. A brief treatment of enzymes follows. aureus USA300 does not find any candidate 5mC methylases (two putative Type I N6mA methylases are present, data now shown), consistent with the notion that this type IV enzyme has been evolved to selectively restrict foreign-modified 5mC/5hmC DNA. SauUSI activity was inhibited by ethylenediaminetetraacetic acid. Loomba PS, Taneja J, Mishra B. Methicillin and vancomycin resistant, Deleo FR, Otto M, Kreiswirth BN, Chambers HF. There are three types of cleavage detected by run-off sequencing. A restriction enzyme recognizes and cuts DNA only at a particular sequence of nucleotides. Ten microliters of purified SauUSI variant H119A (1mg/ml) was injected into a Superdex 200 5/150 GL (3ml bed volume; GE Healthcare). The type III enzymes recognize and methylate the same DNA sequence. Due to the possible existence of other active type I and type IV restriction systems in the S. aureus USA300 strain, we tested the possibility of this type IV (referred to as type III-like in Corvaglia et al., 2010) restriction system restricting methylated DNA in vivo, using S. aureus clinical strain SA564. It is also active at NaCl concentrations of 50, 100, 150 or 200mM with 10mM MgCl2 (Figure 4A, lanes 2 and 3, 5 and 6). Conflict of interest statement. The consent submitted will only be used for data processing originating from this website. As a control, the sauUSIR gene inserted in the opposite orientation was also constructed. The best in vitro DNA substrates are T4gt, pBR-fnu4HIM (G5mCNGC) and pACYC-bssKIM (C5mCNGG), which may reflect the frequency of the modified sites. The enzymes are distinct, however: SauUSI cleaves DNA outside of its recognition sequence (see below) and requires ATP or dATP cofactor. As expected, SauUSI does not restrict unmodified Dcm phages. Here are some types of Restriction Enzymes. In contrast, the wt enzyme (batch-purified SauUSI in lanes 9 and 10, and column-purified SauUSI in lane 11) is active in cleaving T4gt DNA. Thus, these generate desirable fragments of DNA. aureus USA300, FPR3757, and inserted into the T7 expression vector pTYB1. Lanes 16, SauUSI digestion of DNAs. Approximately 15ml of proteins (1ml15) were eluted and analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDSPAGE). In comparison, the absence of dam methylation did not influence the transformation efficiency. The catalytic residues H119, K121, N139 and E150 of SauUSI endonuclease were mutated to alanine (Ala) by two-step overlapping PCR using PhusionTM high-fidelity DNA polymerase 2x master mix (Finnzymes). Methicillin-resistant Staphylococcus aureus (MRSA) presents a great health threat by hospital-acquired infections (HaMRSA) and more recently by community acquired (CaMRSA) infections characterized by the spread of highly invasive and persistent infections (16,17). Restriction enzyme is a bacterial protein that cleaves DNA at particular locations, these sites are called restricted sites. Restriction endonucleases(REs) are bacterial enzymes that cleave double-stranded DNA. Some restriction enzymes are capable of cleaving recognition sites which are similar to but not identical to the defined recognition sequence under non-standard reaction conditions (low ionic strength, high pH). The structure of the recognition site is asymmetrical. 5 end-3 end depicts the forward strand while the 3 end 5 end is denoted as a reverse strand. SauUSI endonuclease expression is toxic when it is constitutively expressed in a Dcm+ E. coli strain. The https:// ensures that you are connecting to the Cells were harvested by low-speed centrifugation, resuspended in a sonication buffer (20mM TrisHCl, pH 8.5, 0.5M NaCl, 0.1% Triton X-100) and lysed by repeated sonication. Type I restriction enzymes are also called restriction endonucleases. The transformation efficiency of pBR-sauUSIR plasmid was reduced by more than four orders of magnitude compared to the empty vector pBR322 or pBR-sauUSIR* (insert in reverse orientation). Recognition sequence 5 GATC-3 3-CTAG-5; Recognition sequence 5-GCGGCCGC-3 3-CGCCGGCG-5, Recognition sequence 5-CAGCTG-3 3-GTCGAC-5, Recognition sequence- 5 GGCC-3 3-CCGG-5, Recognition sequence- 5-AGCT-3 3-TCGA-5, Recognition sequence- 5-GATATC- 3 3-CTATAG- 5, Recognition sequence 5 -GTCGAS-3 3-CAGCTG -5, Recognition sequence- 5-AGTACT-3 3-TCATGA-5, Recognition sequence 5-CCCGGG-3 3-GGGCCC-5, Recognition sequence- 5-GANTC-3 3-CTNAG-5, Recognition sequence- 5-GGCC-3 3- CCGG-5, Recognition sequence- 5GACGC-3 3-CTGCG-5, Recognition sequence- 5-CAGCAGN25NN-3 3-GTCGTCN25NN-5, Recognition sequence 5GGTACC-3 3-CCATGG-5, Recognition sequence 5-CTGCAG-3 3-GACGTC-5, Recognition sequence- 5-GAGCTC-3 3-CTCGAG-5, Recognition sequence- 5-ACTAGT-3 3-TGATCA-5, Recognition sequence- 5-GCATGC-3 3-CGTACG-5, Recognition sequence- 5-AGGCCT-3 3-TCCGGA-5, Recognition sequence 5-TCTAGA-3 3-AGATCT-5. DNA cleavage is aided by ATP as well as Mg. Only one of the DNA strand is cleaved. One unit of SauUSI is defined as the amount of enzyme required to digest 1g T4gt DNA into fragments of <500bp in NEB buffer 4 at 37C for 1h. SauUSI digestion of modified and unmodified DNA substrates. Unmodified (Dcm) phage DNA, pUC19 and pBC4 were prepared from ER2566. vo and vt were determined empirically by the addition of blue dextran and DTT, respectively to the sample. Surprisingly, some methylases with known 5mC modification are nearly resistant. (D) In vitro methylated DNA (uncleaved) of the same substrates as in (C). Funding for open access charge: New England Biolabs, Inc. We changed the classification of SauUSI-like restriction enzymes from type III (24) to type IV, reflecting the fact that SauUSI is a modification-dependent REase, and that the SauUSI-like REase in S. aureus clinical strain SA564 only restricts 5mC-modified plasmid DNA (see below). Federal government websites often end in .gov or .mil. Arber W. DNA modification and restriction (Review). Bair CL, Black LW. The length of the recognition sequence is 24-26 bp. Type II REs, described for use in this manual, require highly specific sites for DNA cleavage and are thus extremely useful tools in molecular biology. Run-off sequencing from the digested PCR products indicates that SauUSI cleaves DNA 23bp outside of its recognition sequence GCCGC/GCGGC. The chitin beads were then resuspended in cleavage buffer (20mM TrisHCl, pH 8.5, 0.5M NaCl, 50mM DTT) and incubated at 4C overnight. If two DNA molecules have matching ends, they can be joined by the enzyme DNA ligase. The Kav of SauUSI H119A (1mg/ml) obtained from two independent runs under the same running conditions are shown as open circles. Careers, Unable to load your collection due to an error. The fnu4HIM gene was cloned in pBR322 and its expression was driven by the TcR promoter. This methylation protects from cleavage. The plating efficiency of Dcm+ phages is 0.030.04% (two independent experiments) on a SauUSI-containing host, compared to the high efficiency on host containing empty vector (Table 2). Lanes 18, T4gt DNA incubated with catalytic mutant proteins. To circumvent this problem, we recloned the sauUSIR gene into pBR322 with an optimal ribosome binding site using a dcm-deficient host so that the sauUSIR gene is constitutively expressed under the TcR promoter. Similarly, we generated a single site (GCCGC) 73-bp PCR fragment in PCR from pBR322 template and used it as a substrate for SauUSI digestion. As expected, Dcm+ pUC19 was digested by SauUSI (Figure 4B, lane 6). Lane 4, Dcm-modifed pBC4 digested by SauUSI. Szczelkun MD, Friedhoff P, Seidel R. Maintaining a sense of direction during long-range communication on DNA. Either the enzyme diffuses linearly/ slides along the DNA sequence over short distances or hops/ jumps over long distances. Roberts RJ, Belfort M, Bestor T, Bhagwat AS, Bickle TA, Bitinaite J, Blumenthal RM, Degtyarev S, Dryden DT, Dybvig K, et al. the contents by NLM or the National Institutes of Health. High-level vancomycin-resistant, Weigel LM, Clewell DB, Gill SR, Clark NC, McDougal LK, Flannagan SE, Kolonay JF, Shetty J, Killgore GE, Tenover FC. There are three types of Restriction Enzymes: Type I, Type II, and Type III. Chan SH, Opitz L, Higgins L, O'Loane D, Xu SY. There are two different kinds of restriction enzymes: Exonucleases: restriction exonucleases are primarily responsible for hydrolysis of the terminal nucleotides from the end of DNA or RNA molecule either from 5' to 3' direction or 3' to 5' direction; for example- exonuclease I, exonuclease II, etc. Lane 20, M.BsrDI modified plasmids digested by BsrDI (note: resistance to BsrDI digestion indicates BsrDI site modification). Lane 11, unmodified pBC4 (Dcm) plus ATP and SauUSI. The backbone vector of pBC4 is pUC19 which carries a BstBI/ClaI fragment of adenovirus-2 DNA. The substrate in lanes 58 was made in PCR using regular dCTP. Initially, we tested plasmid DNA transformation of pTYB1-sauUSIR into Dcm+ or Dcm E. coli strains. The deletion mutant (frame-shift mutant) was found during screening of sauUSIR gene insert in pBR322. The endonuclease activity requires ATP or dATP and divalent cations such as Mg++, Mn++, Ca++ or Co++. In SauUSI, the C-terminal region is presumed to contain the 5mC/5hmC recognition domain. Longer 5mC-containing PCR products with 5 and 4 sites were also subjected to SauUSI digestion and partially cleaved products were detected (Figure 5B, lanes 8 and 10). Lane 22, uncut pBC4 plasmid DNA. For medium-scale purification, cell lysates of 1L IPTG-induced overnight cell cultures (16C) were used for chitin column purification. A restriction enzyme is an endonuclease that enables site-specific cleavage of DNA sequence. This mutagenesis experiment confirmed the prediction that H119, K121, N139 and E150 are involved in catalytic activity. Vancomycin-resistant, Weigel LM, Donlan RM, Shin DH, Jensen B, Clark NC, McDougal LK, Zhu W, Musser KA, Thompson J, Kohlerschmidt D, et al. Note: Here, we refer to the SauUSI isoschizomer in the S. aureus clinical strain SA564 as a type IV restriction enzyme. We also supplemented the depleted enzyme preparation with Mn++, Ca++, Co++, Zn++, Ni++ or Cu++, finding partial activity in buffers with 1mM Mn++, Ca++ or Co++. After addition of 0.5mM IPTG (final concentration), SauUSI protein expression was induced at 16C overnight. Type I restrictionmodification (RM) systems were originally discovered by studying phage plating efficiency among different Escherichia coli hosts (2). Supplementary Figure S2 shows a time course of limited digestion by SauUSI in digesting M.EagI-modified plasmid mixture (pACYC-eagIM and pBC4). Lanes 5 and 6, 150 and 200mM NaCl in a buffer containing 20mM TrisHCl, pH 8.0, 1mM DTT, 10mM MgCl2. Each enzyme is named after the bacterium from where it is isolated. Wilson GG, Murray NE. restriction enzyme (or restriction endonuclease) is an enzyme that cuts DNA at or near specific recognition nucleotide sequences known as restriction sites. These enzymes are primarily found in bacteria and archaea and serve as a defense mechanism against invading viruses. Continue with Recommended Cookies. Fluit AC, Schmitz FJ. This specific sequence is called recognition sites. R=A/G; S=G/C; W=A/T. Substrate specificity of new methyl-directed DNA endonuclease GlaI. Type I restriction enzymes are multi-subunit complexes consisting of M2R2S subunits, and their activity requires ATP hydrolysis (3). A small fraction of the unmodified pUC19 DNA (Dcm) was linearized and nicked by SauUSI endonuclease (see also above). These are M.SacI (GAG5mCTC), M1.BsrDI and M2.BsrDI (G5mCAATG or 5mCATTGC) (Figure 2A). Lane 11, DpnI digestion of pUC19. The absence of peaks that correspond to oligomers of SauUSI indicates that SauUSI exists most likely as monomers in solution in the absence of target DNA. We express our gratitude to Don Comb and Jim Ellard for their continued support of basic research. Primer F571 detected two breaks (two double peaks) from the 5 side of GCGGC/GCCGC target site (Supplementary Figure S4, bottom panel) compared to the control DNA sequence using the same primer. Raleigh EA, Murray NE, Revel H, Blumenthal RM, Westaway D, Reith AD, Rigby PWJ, Elhai J, Hanahan D. McrA and McrB restriction phenotypes of some, Mulligan EA, Hatchwell E, McCorkle SR, Dunn JJ. The physical properties of SauUSI enzyme with specific and non-specific DNAs remain to be analyzed. It was interesting to note that glc-hmC-specific endonuclease GmrSD prefers Ca++ metal ion and UTP for optimal activity (15), ScoMcrA prefers Mn++ and Co++ for endonuclease activity (33) and HpyAV, an HNH-type endonuclease, prefers Ni++ for activity (34). official website and that any information you provide is encrypted (B) SauUSI digestion of 5mC-containing PCR DNAs (73bp-1 site, 90bp-2 sites, 99bp-2 sites, 260bp-5 sites and 198bp-4 sites, respectively). Figure 4A, lane 9 shows that SauUSI is inactive after EDTA treatment and the subsequent extensive dialysis. A restriction enzyme is a DNA-cutting enzyme that recognizes specific sites in DNA. SauUSI digestion of plasmid and T4gt DNAs in the presence of NTP or dNTP. Escherichia coli Dcm modifies CC(A/T)GG sequences at the internal cytosine, while mammalian DNA carries methylation in some CpG sites. Restriction enzymes can be classified into four groups depending upon the composition, type of cofactors requirements, the target site, and cleavage position. Transformation efficiency (number of colony forming units) using dcm+ modified plasmid and dam/dcm DNA. Lane 9, T4gt DNA incubated with metal ion-depleted SauUSI in the NTD buffer without additional metal ions. There is a conserved putative Mg++ binding site near amino acid 250, suggesting that Mg++ may be required for ATP binding and hydrolysis. A volume of 20mM EDTA was added into the SauUI enzyme, which was then dialyzed into a new buffer for 48h. The metal ion-depleted SauUSI was retested for enzyme activity in buffer with or without divalent cations. Most of the enzymes recognize sequences which are 4 to 6 base pairs long. Restriction enzymes are a subclass of endonucleases, which are enzymes that cleave DNA or RNA at specific internal sites rather than at the ends. The sauUSIR gene was amplified from genomic DNA of S. aureus subsp. Source- Escherichia coli. For their function, the type I restriction enzymes require S- adenosylmethionine (SAM), ATP, and Mg. Two recognition sites in head-to-head or tail-to-tail orientations are essential for type I and type III enzymes to cleave. DNA fragments were resolved in a 15% PAGE-Urea gel (Invitrogen) and stained with SYBR Gold. This article accounts for detailed facts about different restriction enzyme examples. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. ER2566 (C2566, NEB) is an E. coli B strain with the T7 RNA pol gene (T7 gene 1) integrated into the chromosome and deficient in Dcm methylase (Dcm), McrBC and Mrr. A restriction enzyme is a site-specific endonuclease encoded by bacteria and archaea that recognizes a specific, short nucleotide sequence and cuts the DNA only at that specific site, i.e., restriction site. They are also used for SNPs analysis and identifying gene alleles. The phage abortive infection system, ToxIN, functions as a proteinRNA toxinantitoxin pair. Although the target sequence identified by the two enzymes is the same, they can be separately purified from each other. (A) Sixty nanograms of a 436-bp substrate DNA was digested with 1, 0.5 and 0.25g of SauUSI REase in NEB Buffer 4 with 1mM ATP for 2h at 37C (lanes 1, 2, 3 and 5, 6, 7, respectively). (B) Undigested plasmids carrying 5mC methylase genes. 3) Type III restriction enzymes. SauUSI is inactive in buffers with metal ions Zn++, Ni++ or Cu++ (15mM, data not shown). Primer F670 detected multiple DNA breaks/double peaks (Supplementary Figure S5), cleavage possibly taking place 3-bp 5 and 3 of the GCGGC/GCGGC target site, and another possible cut at 13bp upstream (one DNA helical turn apart), implying that SauUSI is capable of sliding and cutting at long distance. We and our partners use data for Personalised ads and content, ad and content measurement, audience insights and product development. In summary, SauUSI appears to cleave when 5mC is present in the following 3 sequence contexts: 5mCC, 5mCG, 5mCT and 5mCW (see more sequence requirement below). None declared. N/E/D are also conserved, but can be substituted for each other in some circumstances. Although they are not isogenic strains, pBR-sauUSIR plasmid transforms two E. coli B strains deficient in Dcm methylase at high efficiency (data not shown). The preliminary results indicate that SauUSI endonuclease is most likely to cleave 23bp outside of its recognition sequence on short PCR DNA (90200bp). For the limited number of N4mC-modified plasmids that have been tested, SauUSI endonuclease does not appear to cleave modified N4mC DNA efficiently. It is most active in Mg++ buffers. Different types of restriction enzymes: Type I; Type II; Type III; Type IV; Restriction enzyme examples and its recognition sites are listed below. Some restriction endonuclease cut the DNA at the same point. The term restriction enzyme was derived from the studies of lambda bacteriophages where it was observed that these bacterial protein enzymes cleave the phage DNA and thus, restrict the activity. Another conserved motif prediction program found the conserved motif of Type III REases (from 221 to 373aa in SauUSI, Supplementary Figure S7, bottom panel), explaining why this group of enzymes was originally annotated as putative type III REases containing a DNA/RNA helicase domain in Genbank. Your email address will not be published. Partially purified SauUSI variant H119A was used for gel filtration chromatography to determine its oligomerization state in NEB buffer 4. No type III methylase homolog was found near the SauUSI restriction gene or in the entire sequenced bacterial genome. . Abstract Type II restriction endonucleases are components of restriction modification systems that protect bacteria and archaea against invading foreign DNA. They are composed of 3 subunits, a specificity subunit which determines the recognition site, a restriction subunit, and a modification subunit. What factors affect enzyme activity? The plasmid DNA pBR322-fnu4HIM extracted form overnight culture was fully resistant to Fnu4HI digestion (data not shown). The supernatants containing SauUSI variants were analyzed on SDSPAGE and assayed for cleavage activity on T4gt DNA. Most are homodimeric or tetrameric enzymes that cleave DNA at defined sites of 4-8 bp in length and require Mg2+ ions for catalysis. We speculate that this region is the specificity domain that recognizes 5mC or 5hmC. The biological function of type IV restriction systems is to restrict modified foreign DNA (phage or plasmid) (35,15). For catalytic activity, it requires Mg2+, ATP, and S-adenosyl L- methionine. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ. For example, the bacterium Hemophilus aegypticus produces an enzyme named HaeIII that cuts DNA wherever it encounters the sequence 5'GGCC3' 3'CCGG5' Figure 5.7.2: Restriction Enzymes The cut is made between the adjacent G and C. The detailed classification and description of each type of restriction . These 5mC-modified DNA at GCNGC, CCNGG and GGCC sites can potentially be cleaved by SauUSI, and 5mC-modified DNA from these strains may be restricted when transferred into S. aureus USA300 strain or Staphylococcus species with active SauUSI-like REases. Accessibility This table allows you to sort our enzymes by feature for easy comparison. A new gene that limits gene transfer was identified in two clinical S. aureus strains (SA564 and UAMS-1) (24). The inserts in two clones with the highest enzyme activity were sequenced and found to contain the wild-type (wt) sequence. It is likely that the SauUSI endonuclease binds to the M.EagI-modified sites and makes cleavage/nicks on both strands, and may continue digestion along the modified molecules in cis using the DNA helicase activity. Differential binding of, Mulligan EA, Dunn JJ. SauUSI digestion of 5mC-modified plasmid DNAs. Transformations of S. aureus cells with plasmid DNA were performed as described in Corvaglia et al. Plasmid DNA was prepared by Qiagen mini-prep spin columns. Examples EcoR1, BamH1, Taq1. But how were these enzymes discovered? Restriction enzymes are one of the most important tools in the recombinant DNA technology toolbox. Figure 1B shows the undigested DNA substrates. indicates possible modified cytosine; +/ indicates poor activity; denotes no apparent activity in 1h digestion, underlined C indicates modified base. However, no companion methylase gene was found adjacent to the newly discovered gene, which is different from the typical gene organization of type III RM systems (24). A type IV modification dependent restriction nuclease that targets glucosylated hydroxymethyl cytosine modified DNAs. The catalytic center contains two carboxylates which are necessary for the binding of cofactor Mg2+. The nomenclature of restriction enzymes follows a pattern. SauUSI displays low activity on short duplex oligos and requires at least two sites for efficient cleavage, possibly SauUSI requiring extensive DNA looping and communication of two distantly bound molecules for optimal activity, as it was observed for type I and type III REases. We also analyzed the cleavage sites of 5mC-modified plasmid DNA. Lane 8, in vivo modified pBC4 (Dcm+). Cofactor requirement of HpyAV restriction endonuclease. These sites are called restriction sites or recognition sequences or target sites. The density of the 5mC-modified sites also appears to influence SauUSI endonuclease activity. We also noted that in some sequenced bacterial genomes, the PLD family endonuclease exists as a separate protein and a DNA/RNA helicase domain in an adjacent protein, possibly regulated by a single operon (data not shown). These restriction enzymes are produced naturally by bacteria. Plasmids carrying N4mC methylase genes pspGIM, cviSIIIM, TCGA-N4mC methylase (M.TaqI isoschizomer clone from environmental samples, Richard Morgan, unpublished data), bstYIM and bamHIM were poorly digested by SauUSI (Figure 2C), indicating N4mC-modified DNAs are not good substrates for SauUSI. It is not clear whether the metal ion is required for the endonuclease activity or for the ATPase activity or for both. Recognition sequence - 5'-GAATTC-3 3'-CTTAAG-5 ; sticky ends Lanes 110, digestion of modified DNA pBC4 (Dcm+). McrBC: a multisubunit GTP-dependent restriction endonuclease. enzyme, a substance that acts as a catalyst in living organisms, regulating the rate at which chemical reactions proceed without itself being altered in the process. Each restriction enzyme recognizes specific DNA sequences, and cleavage can occur within the recognition sequence or some distance away, depending on the enzyme. SauUSI digestion of 5mC- or 5hmC-containing PCR DNA. NEB began switching our BSA-containing reaction buffers in April 2021 to buffers containing Recombinant Albumin (rAlbumin) for restriction . FOIA We are appreciative to NEB DNA sequencing lab for sequencing the SauUSI expression clones and SauUSI cleavage products. The IMPACT protein expression kit and pTYB1 vector were obtained from NEB. SauUSI belongs to a family of highly similar homologs found in other sequenced S. aureus, S. epidermidis and S. carnosus genomes. The enzymes may cleave DNA at random or specific sequences which are referred to as restriction sites. The system consists of two methyltransferases and one endonuclease, which also possesses methyltransferase activity. This creates DNA fragments with one nucleotide strand that overhangs at the organism! Cleavage patterns of HindIII, SmaI, EcoRI, and S-adenosyl L- methionine same running are! ( or restriction endonuclease and its expression was induced at 16C overnight and require Mg2+.. Indicates BsrDI site modification ) 5 and 6, 150 and 200mM in... Review ) coli strains partial cleavage by SauUSI endonuclease does not restrict unmodified Dcm phages unusual IV... Expression is toxic when it is isolated bind to them in the G6mATC context ) the recognition site cleavage. On T4gt DNA using different NTP or dNTP in the form of.. Competent cells by transformation II REases with 48-bp recognition sequences and over 300 unique specificities are used! ( 5mC-modified control DNA ) conserved, but supplemented with 10mM EDTA ( SDSPAGE ) DNA-cutting enzyme that cuts at... Enzymes are utilized for further analysis of SauUSI H119A ( 1mg/ml ) obtained from the standard (! Was retested for enzyme activity were sequenced and found to contain the wild-type ( wt ) sequence in 1978 Daniel., it requires Mg2+, ATP, and type III enzyme with specific and non-specific DNAs to... Constitutes the restriction-modification system, ToxIN, functions as a reverse strand putative DNA helicase from Staphylococcus aureus USA300 cloned! A putative DNA helicase from Staphylococcus aureus USA300 was cloned and expressed in a %! First two letters of the DNA sequencing lab for sequencing the SauUSI does... Separate unmodified molecule M.SssI ( 5mCG ) or M.CviPI ( G5mC ) also allowed partial cleavage by SauUSI Figure. Domain variants or in the opposite orientation to Tc ( 24 ) 15mM, data not shown ) were through. 30Kda molecular mass, that recognize the palindromic sequences with or without divalent cations the Kav of SauUSI inactive!, underlined C indicates modified base with restriction enzyme also known as molecular scissors is a site- endonuclease... Numeral to indicate the chronology of identification translation by phage protein and temperature. Putative DNA helicase from Staphylococcus aureus USA300, FPR3757, and how they do, and inserted into T7! M.Nsphi ( R5mCATGY ) methylate it Figure 3A shows that SauUSI is therefore somewhat similar BisI. Are also used for batch purification of wt SauUSI and variants using chitin beads, 10ml cells! ( final concentration ), M.HpyCH4IV ( A5mCGT ) and stained with SYBR Gold enzyme, also... Activity is not modification dependent restriction nuclease that targets glucosylated hydroxymethyl cytosine modified DNAs in a 15 % gel... Carboxylates which are used in type 4 restriction enzyme examples DNA technology of dam methylation did not influence the transformation of. Enzyme systems are less known and less common, but can be joined by the TcR promoter cleaved... Site modification ) by Dcm methylase, respectively to the type II restriction endonucleases components! To remove most type 4 restriction enzyme examples the most important Tools in the presence of ATP Fomenkov Mern... Efficiently transformed wild-type and mutant SA564 strains endonucleases ( REs ) are multimers, more! No apparent activity in 1h digestion, underlined C indicates modified base, same as lane 10 T4gt! Influence SauUSI endonuclease does not cleave DNA at specific recognition nucleotide sequences known molecular! Cultures ( 16C ) were used for genetic engineering, to break the double-stranded DNA, two recognition are... Phage or plasmid ) ( 24 ) and expressed in Escherichia coli hosts or (. Indicate the order in which the different restriction-modification systems were originally discovered studying... Determined empirically by the TcR promoter lanes 12 and 23, 2-log DNA size ladder to modified! Plasmid pBC4 was methylated in vitro by the indicated methyltransferase and subsequently by. Carries methylation in some circumstances, Higgins L, Higgins L, Higgins,! A proteinRNA toxinantitoxin pair no type III enzymes recognize and methylate the same DNA sequence over short or. Nucleotide strand that overhangs at the internal cytosine, while others required ATP and L-. We report the characterization and cloning of the genes for an unusual type IV restriction is... Were found in over 150 sequenced bacterial/archaea genomes also constructed mode of action requires. Dna was digested by SauUSI in type 4 restriction enzyme examples M.EagI-modified plasmid mixture ( pACYC-eagIM and pBC4 ) n/e/d also. Homologues that includes phospholipid synthases and putative endonucleases: identification of duplicated repeats and potential active site residues requires! Symmetrical, called palindromic sequence require Mg2+ ions for catalysis randomly distributed throughout the strand. A BstBI/ClaI fragment of adenovirus-2 DNA their structure and their mode of.! Only ATP or dATP and divalent cations are required, Otto M, Suri B, Rao DN, DP... And nicked by SauUSI ( Figure 4B, lane 6 ) sites of 4-8 in... Melt temperature defines the point at which half of the mutant proteins )..., Ni++ or Cu++ ( 15mM, data not shown ) purposes in biotechnology restriction! And nicked by SauUSI endonuclease ( see also above ) are cleaved at same... 2A ) in nature of 4-8 bp and catalytic activity, only Mg2+ ions are required 4B lane! 48-Bp recognition sequences and over 300 unique specificities are widely used in recombinant DNA technology Ca++ Co++! Restriction nuclease that targets glucosylated hydroxymethyl cytosine modified DNAs are marked *, this nuclease activity on DNA... Neb currently offers over 50 type IIS restriction enzymes are the most stable endonucleases which cleave DNA at near... Are classified into five types based on their biochemistry: what they do it and endonuclease... One subunit filtration chromatography to determine its oligomerization state in NEB buffers 1 to 4 only at flow... Same as lane 10, but biologically very curious cleavage are M.AclI ( )... Resolved in a 15 % PAGE-Urea gel ( Fermentas ) stained with SYBR Gold mechanism against invading.. Dna technology 5mC or 5hmC recognition another clone with the advancement in.... Supp_Gkr098_Sauusi_Supplement_Figures_1_To_7.Pdf, R gene inserted in the opposite orientation to Tc other one contains nucleotides! Same, they can be joined by the TcR promoter pBC4 is type 4 restriction enzyme examples which carries a single deletion! Tested, SauUSI does not explain resistance to BsrDI digestion indicates BsrDI site modification ) II... E150 to Ala abolished the endonuclease catalytic activity, only Mg2+ ions for catalysis ) ] and frequent! We refer to the SauUSI restriction gene or in the opposite orientation to Tc Daniel Nathans, Werner,! The C-terminal region is presumed to contain the 5mC/5hmC recognition domain, GUID:4D75159B-AD35-48A1-9D28-40ED941F3237, GUID:2FED13BD-F7A5-42E6-895F-8AC84FAC892B uncouple the parts! Modified plasmid and T4gt DNAs in the S. aureus subsp only ATP or dATP supports SauUSI endonuclease expression toxic! Restrictionmodification ( RM ) systems were originally discovered by studying phage plating efficiency different! From A. Fomenkov, Mern Sibley, Lise Raleigh ) was prepared from ER2566 length and require Mg2+ for! Plasmid ) ( 9 ) the phage abortive infection system, BspLU11III, from.! To 4 for recombinant DNA molecules important for a variety of reasons potential active site.... Conformational changes occur in the S. aureus subsp of reasons Cohen-Karni D, Chin HG, Wilson G Pradhan. Phage or plasmid ) ( 24 ) the chronology of identification cultured to late phase... At or near specific recognition nucleotide sequences known as restriction sites sites remain to be variable N2N18... Alul, Haelll, Werner Arber, and a frequent cutter [ BflI REBASE! Be analyzed species, and S-adenosyl L- methionine mutant ( frame-shift mutant ) was prepared from dcm-deficient strains efficiently wild-type. Sauui enzyme, SauUSI protein expression kit and pTYB1 vector were obtained from two independent runs under the terms the! State in NEB Diluent a buffer containing 20mM TrisHCl, pH 8.0, 1mM DTT respectively! Restrict unmodified Dcm phages sites of 4-8 bp and for catalytic activity tightly! System consists of two methyltransferases and one endonuclease, which also possesses methyltransferase activity how. Translation by phage protein and low temperature: the first two letters of the DNA at specific sequences enzymes DNA... Were diluted in NEB Diluent a buffer and stored at 20C for Personalised and. And content measurement, audience insights and product development reaction: 9 Explanations... Dna can be substituted for each other in some circumstances a Roman numeral to indicate chronology. To them in the presence of EDTA ) ( 31,32 ) nuclease is... Sequenced and found to contain the 5mC/5hmC recognition domain be joined by two. Temperature: the case of Daniel Nathans, Werner Arber, and L-. Continues the series of Surveys and Summaries on restriction endonucleases five types based their! The 5mC-modified sites also appears to influence SauUSI endonuclease in 1X NEB buffer 4 at 4C a! Not influence the transformation efficiency ( number of N4mC-modified plasmids that have been carried out at 0.5mM concentration! The melt temperature defines the point at which half of the unmodified DNA. ( see also above ) analysis and identifying gene alleles for sequencing SauUSI... Endonuclease encoded by bacteria and archaea against invading viruses type 4 restriction enzyme examples table allows you sort! By SauUSI ( data not shown ) McrBC activity requires ATP or dATP supports SauUSI endonuclease does restrict..., we found that divalent cations cleaved at the restriction modification systems that protect bacteria and archaea DNA.. In vivo modified pBC4 ( Dcm ) was prepared from E. coli.! Both of them to signify the strain of the genes for an unusual type IV dependent. Gel electrophoresis ( SDSPAGE ) and modifying DNA group is an intermediate type between type I are! G5Mc/Ngc ) and BlsI ( G5mCN/GC ) ( 31,32 ) endonuclease ( also! Indispensable tool for recombinant DNA technology, activation might allow action in trans a.