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Research Article Lrai from Lactococcus Raffinolactis BGTRK10-1, an Isoschizomer of Ecori, Exhibits Ion Concentration-Dependent Specific Star Activity

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Research Article Lrai from Lactococcus Raffinolactis BGTRK10-1, an Isoschizomer of Ecori, Exhibits Ion Concentration-Dependent Specific Star Activity Hindawi BioMed Research International Volume 2018, Article ID 5657085, 10 pages https://doi.org/10.1155/2018/5657085 Research Article LraI from Lactococcus raffinolactis BGTRK10-1, an Isoschizomer of EcoRI, Exhibits Ion Concentration-Dependent Specific Star Activity Marija Miljkovic,1 Milka Malesevic,1 Brankica Filipic,1,2 Goran Vukotic,1,3 and Milan Kojic 1 1 Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Belgrade, Serbia 2Faculty of Pharmacy, University of Belgrade, Belgrade, Serbia 3Faculty of Biology, University of Belgrade, Belgrade, Serbia Correspondence should be addressed to Milan Kojic; [email protected] Received 22 November 2017; Accepted 2 January 2018; Published 29 January 2018 Academic Editor: Gamal Enan Copyright © 2018 Marija Miljkovic et al. Tis is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Restriction enzymes are the main defence system against foreign DNA, in charge of preserving genome integrity. Lactococcus rafnolactis BGTRK10-1 expresses LraI Type II restriction-modifcation enzyme, whose activity is similar to that shown for EcoRI; LraI methyltransferase protects DNA from EcoRI cleavage. Te gene encoding LraI endonuclease was cloned and overexpressed ∘ ∘ in E. coli. Purifed enzyme showed the highest specifc activity at lower temperatures (between 13 Cand37C) and was stable afer ∘ storage at −20 C in 50% glycerol. Te concentration of monovalent ions in the reaction bufer required for optimal activity of LraI restriction enzyme was 100 mM or higher. Te recognition and cleavage sequence for LraI restriction enzyme was determined � � as 5 -G/AATTC-3 , indicating that LraI restriction enzyme is an isoschizomer of EcoRI. In the reaction bufer with a lower salt � � concentration, LraI exhibits star activity and specifcally recognizes and cuts another alternative sequence 5 -A/AATTC-3 ,leaving the same sticky ends on fragments as EcoRI, which makes them clonable into a linearized vector. Phylogenetic analysis based on sequence alignment pointed out the common origin of LraI restriction-modifcation system with previously described EcoRI-like restriction-modifcation systems. 1. Introduction their subunit composition, cofactor requirement, recognition site, cleavage site, and mode of action to defne the diferent Restriction endonucleases are generally accompanied by a cog- types (I, II, III, and IV). Restriction endonucleases Type II nate methyltransferase [1]. Both enzymes working together form are essential tools for recombinant DNA technology. It seems a restriction-modifcation system (RM system). RM systems unlikely that today’s modern molecular biology and the are important for the maintenance of the genome integrity biotechnologyindustrywouldhavedevelopedwithoutType of prokaryotic organisms. Te range of biological processes II restriction enzymes. Because of their great importance that utilize RM system also includes involvement in DNA in gene analysis and cloning there is a constant need to transposition [2] and recombination [3]. In addition, there is discover new ones. According to data from the REBASE evidence that the genes for restriction and modifcation en- [6, http://rebase.neb.com] which summarizes all information zymes may act together as selfsh elements [4, 5]. known about every restriction enzyme and any associated Restriction endonucleases exhibit high sequence speci- protein, there are more than 3945 biochemically or geneti- fcity in substrate binding and use versatile DNA cleavage cally characterized restriction enzymes and, out of 3834 Type mechanisms and thus are excellent model systems for under- II restriction enzymes, 299 distinct specifcities are known. By standing DNA recognition and phosphodiester bond hydrol- 2010, six hundred and forty-one restriction enzymes were ysis. Restriction endonucleases are classifed according to commercially available, including 235 distinct specifcities [7]. 2 BioMed Research International ∘ Because of the large number of sequenced genomes, rate of Montelieu-Vercieu, France), temperature of growth (30 C, ∘ ∘ discovery of new putative restriction and modifcation genes 37 C, and 45 C), growth in the presence of salt (4% and is rising rapidly. In contrast, the number of restriction en- 6.5%), and pH tolerance. Final taxonomic classifcation of zymes that are biochemically characterized has actually BGTRK10-1 was performed by sequencing of amplifed 16S dropped down to the level that was three decades ago. rDNA using primers previously described [18]. Te strain was Restriction endonucleases Type II are homodimeric or grown in M17 medium (Merck GmbH, Darmstadt, Germany) ∘ tetrameric enzymes that cleave DNA at defned sites of 4–8 bp supplemented with D-glucose (0.5% w/v) (GM17) at 30 C. 2+ Escherichia coli DH5�, HB101, and ER2523 strains were in length and require Mg ions for catalysis [8]. For many of ∘ restriction endonucleases Type II, it was found that modifed grown aerobically in Luria-Bertani (LB) broth at 37 C, unless conditions (lower ionic strength, higher pH, presence of otherwise specifed. Solid medium was made by adding diferent metallic cofactors, and organic solvents) could 1.75% (w/v) agar (Torlak, Belgrade, Serbia), to the liquid decrease their substrate specifcity [9–13]. Under nonoptimal media. Antibiotics were used at the following concentra- restriction conditions, these endonucleases can usually cleave tions: erythromycin 300 �g/ml and ampicillin 100 �g/ml for degenerate sequences, which difer from standard recogni- selection and maintaining of transformants. Te 5-bromo-4- tion sites at only one nucleotide. Tis alteration in digestion chloro-3-indolyl-�-D-galacto-pyranoside (X-Gal) (Fermen- specifcity causing cleavage of DNA at novel, similar but not tas, Vilnius, Lithuania) was added to LB medium plates identical sequences is defned as enzyme star activity. Mod- for blue/white colour screening of colonies with cloned ifed specifcity of restriction enzyme (star) activity could fragments at fnal concentration of 40 �g/ml. be exploited to facilitate recombinant DNA techniques since thesameenzymeincontrolledconditionscouldrecognize 2.2. Construction of Cosmid Library of L. rafnolactis diferent DNA sequences and cleave at additional positions BGTRK10-1. Total DNA isolated from the L. rafnolactis [9]. BGTRK10-1 was partially digested with XbaI restriction Restriction endonucleases with identical recognition sites enzyme. Incubation was carried out during 1 h and was isolated from diferent organisms are termed isoschizomers stopped in diferent time intervals by adding EDTA. Opti- mally digested DNA, giving fragments 30–40 kb, was purifed [7, 14]. Te RsrI endonuclease found in Rhodobacter sphae- ∘ roides is an isoschizomer of the EcoRI. Both enzymes recog- and ligated overnight at 16 CwiththepAZILcosvector[19] nizethesequenceGAATTCandcleaveitatthesameposition predigested with XbaI restriction enzyme and dephospho- (G/AATTC) and are sharing 50% amino acid sequence rylated. Ligation for formed concatemers of high molecular identity [15]. Interesting, MunIrecognizesthesequence weight was checked on agarose gel and encapsulated into CAATTG, which difers from the recognition sequence of phage particles using packaging kit (Agilent Technologies). EcoRI (and RsrI) only in the external base pairs. Comparison Encapsulated cosmids were transfected into E. coli HB101 of the MunI amino acid sequence with that of EcoRIand magnesium cells and selection of clones was done on LA RsrI revealed only a low level of overall similarity wherefore plates containing erythromycin 300 �g/ml. Constructed cos- mid library in E. coli was stored in LB containing 15% (v/v) sequence homology between EcoRI and RsrIhasastronger ∘ signifcance [16]. glycerol at −80 C. Tis work describes for the frst time the occurrence of EcoRI-like restriction-modifcation genes in lactococci. Te 2.3. DNA Manipulations. Total DNA from L. rafnolactis objective was to clone, purify, and biochemically and genet- BGTRK10-1 was isolated by modifed method described by ically characterize novel lactococcal LraI restriction enzyme. Hopwood et al. [20]; the logarithmic phase cells were ∘ LraI restriction enzyme was overexpressed and purifed to pretreated with lysozyme (4 mg/ml, for 15 min at 37 C) prior the homogeneity from E. coli using pMAL expression and to treatment with SDS. For plasmid isolation from E. coli the purifcation system. Results demonstrate that LraI restriction QIAprep Spin Miniprep kit was used according to the manu- enzyme, although an isoschizomer of EcoRI, shows diferent facturer’s recommendations (Qiagen, Hilden, Germany). characteristics. One of characteristics that could be further Standard heat-shock transformation was used for plasmid exploited is star activity of LraIthatislimitedtoonevariant transfer into E. coli [21]. Digestion with restriction enzymes of the recognition site, which afer cleavage leaves identical was conducted according to the supplier’s instructions (Ter- cohesive ends as EcoRI and LraI restriction enzymes, so that mo Fisher Scientifc). Te DNA fragments from agarose gels the fragments obtained afer digestion could be cloned with- were purifed using QIAqick Gel extraction kit as described out additional processing. by the manufacturer (Qiagen, Hilden, Germany). DNA was ligated with T4 DNA ligase (Agilent technologies, USA) ac- 2. Material and Methods cording to
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