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Molecular cloning of the three base restriction endonuclease R.CviJI from eukaryotic Chlorella virus IL-3A.

ABSTRACT: R.CviJI is unique among site-specific restriction endonucleases in that its activity can be modulated to recognize either a two or three base sequence. Normally R.CviJI cleaves RGCY sites between the G and C to leave blunt ends. In the presence of ATP R.CviJI* cleaves RGCN and YGCY sites, but not YGCR sites. The gene encoding R.CviJI was cloned from the eukaryotic Chlorella virus IL-3A and expressed in Escherichia coli. The primary E.coli cviJIR gene product is a 278 amino acid protein initiated from a GTG codon, rather than the expected 358 amino acid protein initiated from an in-frame upstream ATG codon. Interestingly, the 278 amino acid protein displays the normal restriction activity but not the R.CviJI* activity of the native enzyme. Nine restriction and modification proteins which recognize a central GC or CG sequence share short regions of identity with R.CviJI amino acids 144-235, suggesting that this region is the recognition and/or catalytic domain.

SUBMITTER: Swaminathan N

PROVIDER: S-EPMC145972 | biostudies-other | 1996 Jul

REPOSITORIES: biostudies-other

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Molecular cloning of the three base restriction endonuclease R.CviJI from eukaryotic Chlorella virus IL-3A.

Swaminathan N N Mead D A DA McMaster K K George D D Van Etten J L JL Skowron P M PM

Nucleic acids research 19960701 13

R.CviJI is unique among site-specific restriction endonucleases in that its activity can be modulated to recognize either a two or three base sequence. Normally R.CviJI cleaves RGCY sites between the G and C to leave blunt ends. In the presence of ATP R.CviJI* cleaves RGCN and YGCY sites, but not YGCR sites. The gene encoding R.CviJI was cloned from the eukaryotic Chlorella virus IL-3A and expressed in Escherichia coli. The primary E.coli cviJIR gene product is a 278 amino acid protein initiated ...[more]

PMID: 8692682

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Project description:BACKGROUND: In continuing our research into the new family of bifunctional restriction endonucleases (REases), we describe the cloning of the tsoIRM gene. Currently, the family includes six thermostable enzymes: TaqII, Tth111II, TthHB27I, TspGWI, TspDTI, TsoI, isolated from various Thermus sp. and two thermolabile enzymes: RpaI and CchII, isolated from mesophilic bacteria Rhodopseudomonas palustris and Chlorobium chlorochromatii, respectively. The enzymes have several properties in common. They are large proteins (molecular size app. 120 kDa), coded by fused genes, with the REase and methyltransferase (MTase) in a single polypeptide, where both activities are affected by S-adenosylmethionine (SAM). They recognize similar asymmetric cognate sites and cleave at a distance of 11/9 nt from the recognition site. Thus far, we have cloned and characterised TaqII, Tth111II, TthHB27I, TspGWI and TspDTI. RESULTS: TsoI REase, which originate from thermophilic Thermus scotoductus RFL4 (T. scotoductus), was cloned in Escherichia coli (E. coli) using two rounds of biochemical selection of the T. scotoductus genomic library for the TsoI methylation phenotype. DNA sequencing of restriction-resistant clones revealed the common open reading frame (ORF) of 3348 bp, coding for a large polypeptide of 1116 aminoacid (aa) residues, which exhibited a high level of similarity to Tth111II (50% identity, 60% similarity). The ORF was PCR-amplified, subcloned into a pET21 derivative under the control of a T7 promoter and was subjected to the third round of biochemical selection in order to isolate error-free clones. Induction experiments resulted in synthesis of an app. 125 kDa protein, exhibiting TsoI-specific DNA cleavage. Also, the wild-type (wt) protein was purified and reaction optima were determined. CONCLUSIONS: Previously we identified and cloned the Thermus family RM genes using a specially developed method based on partial proteolysis of thermostable REases. In the case of TsoI the classic biochemical selection method was successful, probably because of the substantially lower optimal reaction temperature of TsoI (app. 10-15°C). That allowed for sufficient MTase activity in vivo in recombinant E. coli. Interestingly, TsoI originates from bacteria with a high optimum growth temperature of 67°C, which indicates that not all bacterial enzymes match an organism's thermophilic nature, and yet remain functional cell components. Besides basic research advances, the cloning and characterisation of the new prototype REase from the Thermus sp. family enzymes is also of practical importance in gene manipulation technology, as it extends the range of available DNA cleavage specificities.

Project description:Prokaryotes possess numerous diverse defense systems to resist viral infections, while some viruses have also evolved antiviral defense systems to exclude other viruses in cases of multiple infections. Here, we report the first virus-derived modification-dependent restriction endonuclease (HHPV4I) from the archaeal virus HHPV4 (Haloarcula hispanica pleomorphic virus 4). HHPV4I contains an SRA domain, a winged helix (wH) domain, and an HNH domain; recognizes the Gm6ATC site; and specifically binds to Gm6ATC site-containing DNA. Both the wH domain and the HNH domain are responsible for DNA binding. Unlike the well-known m6A-specific restriction enzyme DpnI, HHPV4I only efficiently cleaves DNA with a fully methylated Gm6ATC site and cleaves DNA both upstream and downstream of the Gm6ATC sites on both DNA strands. Furthermore, HHPV4I preferentially cleaves DNA between VR bases (V = A/G/C, R = A/G) 4 to 20 nt away from the Gm6ATC site. Thus, the cleavage pattern of HHPV4I is distinct from those of all of the presently characterized restriction endonucleases. Mutations in the wH domain of HHPV4I do not alter m6A-dependent endonuclease activity, but they decrease recognition sequence specificity, thus expanding the cleaving capacity to more m6A-containing DNA sequences. The wH domain provides a target for searching, developing, and engineering novel m6A-dependent endonucleases. IMPORTANCE Many modification-dependent restriction endonucleases (MDREs) were identified in prokaryotes and recognized modified cytosine bases, such as 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), and glucosyl-5-hydroxymethylcytosine (g5hmC). The first virus-derived MDRE (HHPV4I) from the archaeal virus HHPV4 was identified in this study. The viral MDRE suggested a new strategy employed by the virus to exclude other viruses in the case of multiple replications. HHPV4I is a novel N6-methyladenine (m6A)-dependent restriction endonuclease, while the cleavage pattern of HHPV4I is distinct from the well-known m6A-dependent restriction endonuclease DpnI. HHPV4I recognizes Gm6ATC sites and cleaves DNA both upstream and downstream of the Gm6ATC sites on both DNA strands. It preferentially cleaves DNA between VR bases (V = A/G/C, R = A/G) 4 to 20 nt away from the Gm6ATC sites. Furthermore, mutations in the HHPV4I wH domain can alter the sequence specificity without impeding the m6A-dependent DNA cleavage activity, providing a target for engineering more m6A-dependent endonucleases with different sequence specificities.

Project description:BackgroundRestriction-modification systems are a diverse class of enzymes. They are classified into four major types: I, II, III and IV. We have previously proposed the existence of a Thermus sp. enzyme family, which belongs to type II restriction endonucleases (REases), however, it features also some characteristics of types I and III. Members include related thermophilic endonucleases: TspGWI, TaqII, TspDTI, and Tth111II.ResultsHere we describe cloning, mutagenesis and analysis of the prototype TspGWI enzyme that recognises the 5'-ACGGA-3' site and cleaves 11/9 nt downstream. We cloned, expressed, and mutagenised the tspgwi gene and investigated the properties of its product, the bifunctional TspGWI restriction/modification enzyme. Since TspGWI does not cleave DNA completely, a cloning method was devised, based on amino acid sequencing of internal proteolytic fragments. The deduced amino acid sequence of the enzyme shares significant sequence similarity with another representative of the Thermus sp. family - TaqII. Interestingly, these enzymes recognise similar, yet different sequences in the DNA. Both enzymes cleave DNA at the same distance, but differ in their ability to cleave single sites and in the requirement of S-adenosylmethionine as an allosteric activator for cleavage. Both the restriction endonuclease (REase) and methyltransferase (MTase) activities of wild type (wt) TspGWI (either recombinant or isolated from Thermus sp.) are dependent on the presence of divalent cations.ConclusionTspGWI is a bifunctional protein comprising a tandem arrangement of Type I-like domains; particularly noticeable is the central HsdM-like module comprising a helical domain and a highly conserved S-adenosylmethionine-binding/catalytic MTase domain, containing DPAVGTG and NPPY motifs. TspGWI also possesses an N-terminal PD-(D/E)XK nuclease domain related to the corresponding domains in HsdR subunits, but lacks the ATP-dependent translocase module of the HsdR subunit and the additional domains that are involved in subunit-subunit interactions in Type I systems. The MTase and REase activities of TspGWI are autonomous and can be uncoupled. Structurally and functionally, the TspGWI protomer appears to be a streamlined 'half' of a Type I enzyme.

Dataset Information

Molecular cloning of the three base restriction endonuclease R.CviJI from eukaryotic Chlorella virus IL-3A.

Publications

Molecular cloning of the three base restriction endonuclease R.CviJI from eukaryotic Chlorella virus IL-3A.

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