A comparison of DNA cleavage by the restriction enzymes SalPI and PstI.

Methods for obtaining highly active, exonuclease-free, stable preparations of the Streptomyces albus P restriction enzyme SalPI are described. SalPI and its isoschizomer PstI (from the taxonomically distant Providencia stuartii 164) both cleave their recognition sequence (5'-CTGCAG-3') to generate fragments terminating in tetranucleotide 3' extensions whose sequence is 5'-TGCA-3'. Bacteriophage R4G2 DNA, protected against SalPI cleavage by pregrowth on S. albus P, is also protected against PstI cleavage; and total DNA of both S. albus P and P. stuartii 164 is resistant to cleavage by both enzymes.     

Transformation of restriction endonuclease phenotype in Streptococcus pneumoniae

The genetic basis of the unique restriction endonuclease DpnI, that cleaves only at a methylated sequence, 5'-GmeATC-3', and of the complementary endonuclease DpnII, which cleaves at the same sequence when it is not methylated, was investigated. Different strains of Streptococcus pneumoniae isolated from patients contained either DpnI (two isolates) or DpnII (six isolates). The latter strains also contained DNA methylated at the 5'-GATC-3' sequence. A restrictable bacteriophage, HB-3, was used to characterize the various strains and to select for transformants. One laboratory strain contained neither DpnI nor Dpn II. It was probably derived from a DpnI-containing strain, and its DNA was not methylated at 5'-GATC-3'. Cells of this strain were transformed to the DpnI restriction phenotype by DNA from a DpnI-containing strain and to the DpnII restriction phenotype by DNA from a DpnII-containing strain. Neither cross-transformation, that is, transformation to one phenotype by DNA from a strain of the other phenotype, nor spontaneous conversion was observed. Extracts of transformants to the new restriction phenotype were shown to contain the corresponding endonuclease.     

Restriction enzyme BstZ17I is sensitive to cytosine methylation

The cleavage patterns of a subset of restriction enzymes are blocked or impaired when a methylated CpG is overlapped with either the 5' or 3' end of the canonical restriction site. BstZ17I restriction endonuclease is a blunt-end cutter, which recognises the hexanucleotide sequence GTA(downward arrow)TAC. In this report, I show that the BstZ17I restriction enzyme is sensitive to cytosine methylation. Using both in vitro-methylated episomal plasmids and lambdaDNA, I demonstrate that the BstZ17I restriction enzyme is sensitive to cytosine methylation that occurs 3' and/or 5' of the canonical recognition sequence.     

Isolation and computer-aided characterization of MmeI, a type II restriction endonuclease from Methylophilus methylotrophus.

A Type II restriction endonuclease, MmeI, has been purified from the obligate methylotroph, Methylophilus methylotrophus. The enzyme was shown to have the non-palindromic recognition sequence 5'-T C C Pu A C (N)20-3', 3'-A G G Py T G (N)18-5' and to cleave (as indicated) on the 3' side, generating a two nucleotide 3' projection. Determination of the recognition sequence was achieved using two new computer programs; RECOG, which predicts recognition sequences from the pattern of restriction fragments obtained from DNAs of known sequence, and GELSIM, which generates graphical simulations of DNA band patterns obtained by gel electrophoresis of restriction digests of sequenced DNA molecules.     

A restriction enzyme from Fusobacterium nucleatum 4H which recognizes GCNGC.

A site-specific restriction endonuclease Fnu4H I isolated from Fusobacterium nucleatum 4H recognizes the DNA nucleotide sequence 5'G C N G C-3'/3'-C G N C G-5' and cleaves as indicated by the arrows.     

BfiI, a restriction endonuclease from Bacillus firmus S8120, which recognizes the novel non-palindromic sequence 5'-ACTGGG(N)5/4-3'.

A new type IIS restriction endonuclease Bfi I hasbeen partially purified from Bacillus firmus S8120. Bfi I recognizes the non-palindromic hexanucleotide sequence 5'-ACTGGG(N)5/4-3' and makes a staggered cut at the fifth base pair downstream of the recognition sequence on the upper strand, producing a single base 3' protruding end.     

Characterization of BseMII, a new type IV restriction-modification system, which recognizes the pentanucleotide sequence 5'-CTCAG(N)(10/8)/

We report the properties of the new BseMII restriction and modification enzymes from Bacillus stearothermophilus Isl 15-111, which recognize the 5'-CTCAG sequence, and the nucleotide sequence of the genes encoding them. The restriction endonuclease R.BseMII makes a staggered cut at the tenth base pair downstream of the recognition sequence on the upper strand, producing a two base 3'-protruding end. Magnesium ions and S:-adenosyl-L-methionine (AdoMet) are required for cleavage. S:-adenosylhomocysteine and sinefungin can replace AdoMet in the cleavage reaction. The BseMII methyltransferase modifies unique adenine residues in both strands of the target sequence 5'-CTCAG-3'/5'-CTGAG-3'. Monomeric R.BseMII in addition to endonucleolytic activity also possesses methyltransferase activity that modifies the A base only within the 5'-CTCAG strand of the target duplex. The deduced amino acid sequence of the restriction endonuclease contains conserved motifs of DNA N6-adenine methylases involved in S-adenosyl-L-methionine binding and catalysis. According to its structure and enzymatic properties, R.BseMII may be regarded as a representative of the type IV restriction endonucleases.     

Characterization of a restriction-modification system of the thermotolerant methylotroph Bacillus methanolicus.

We report the isolation of a restriction endonuclease, BmeTI, an isoschizomer of BclI, that recognizes the DNA sequence 5' TGATCA 3'. We also report that BmeTI sites are modified to TGm6ATCA. These findings provide the basis for devising strategies to prevent BmeTI restriction of any DNA introduced into Bacillus methanolicus.     

Site-specific restriction endonuclease RME211 from Rhizobium meliloti]

A new site-specific restriction endonuclease Rme211 from Rhizobium meliloti has been shown to recognize the following nucleotide sequence 5'-ATCGAT-3' in the double-stranded DNA. Thus, the enzyme is a true isoschizomer for restriction endonucleases Bsu151 and ClaI.     

Electron microscopy studies of DNA complexes with restriction endonuclease SalGI

The binding properties of the type II restriction endonuclease SalGI to the plasmid DNA pGW 10 has been investigated by electron microscopic studies. Samples were spread by the BAC technique. In the presence of magnesium, SalGI binds as dimers and tetramers to the specific recognition site 5'-G-T-C-G-A-C-3' and with lower rate to the sequence 5'-G-T-C-A-A-C-3', which represents the recognition site of the restriction endonucleases Hind II and Hinc II.     

A new endonuclease recognizing the deoxynucleotide sequence CCNNGG from the cyanobacterium Synechocystis 6701.

A new sequence-specific endonuclease from the cyanobacterium Synechocystis species PCC 6701 has been purified and characterized. This enzyme, SecI, is unique in recognizing the nucleotide sequence: 5' -CCNNGG-3' 3' -GGNNCC-5' and cleaves it at the position indicated by the symbol. Two other restriction endonucleases, SecII and SecIII, found in this organism are isoschizomers of MspI and MstII, respectively.     

Some restriction endonucleases tolerate single mismatches of the pyrimidine.purine type.

DNAs from phage mutants M13mp18 and M13mp18/MP-1 were used to construct two closed circular heteroduplexes. One of them carried the sequence 5'-CCTGGG-3' 3'-GGGCCC-5' with a T.G mismatch at the position 6248. The other carried the sequence 5'-CCCGGG-3' 3'-GGACCC-5' with a C.A mismatch at the same position. Heteroduplexes were exposed to 7 restriction endonucleases having recognition sites within the sequence 5'-CCCGGG-3' 3'-GGGCCC-5' and to 1 restriction endonuclease having a recognition site within the sequence 5'-CCTGGG-3' 3'-GGACCC-5'. All tested enzymes cleaved at least one mismatch-containing sequence although with reduced efficiency. Smal and Xmal tolerated both mismatch-containing sequences. Aval, Hpall, Mspl, Ncil and Nsplll were able to tolerate only the T.G containing sequence, while BstNl was able to tolerate only the C.A containing sequence. It is inferred that the tolerance displayed by Smal and Xmal depends on the presence of either the original purines or the original pyrimidines in mismatches of both the T.G and C.A type and that all other tested enzymes require the presence of the original purines in mismaches of both types.     

Characterization of a methyl-specific restriction system in Clostridium acetobutylicum strain N1-4081

A type II restriction endonuclease, named CacI, was detected in Clostridium acetobutylicum strain N1-4081. CacI cleaved the tetranucleotide sequence [5' decreases -GATC-3']. The modification system consisted of the methylation of the adenine present in this sequence. CacI, an isoschizomer of MboI, is inactive on dam methylated substrates.     

PfoI,a unique type II restriction endonuclease that recognises the sequence 5'-T downward arrow CCNGGA-3'

A new type II restriction endonuclease designated PfoI has been partially purified from Pseudomonas fluorescens biovar 126. PfoI recognises the interrupted hexanucleotide palindromic sequence 5'-T downward arrow CCNGGA-3' and cleaves DNA to produce protruding pentanucleotide 5'-ends.     

A new Thermus sp. class-IIS enzyme sub-family: isolation of a 'twin' endonuclease TspDTI with a novel specificity 5'-ATGAA(N(11/9))-3', related to TspGWI,TaqII and Tth111II

The TspDTI restriction endonuclease, which shows a novel recognition specificity 5'-ATGAA(N(11/9))-3', was isolated from Thermus sp. DT. TspDTI appears to be a 'twin' of restriction endonuclease TspGWI from Thermus sp. GW, as we have previously reported. TspGWI was isolated from the same location as TspDTI, it recognizes a related sequence 5'-ACGGA(N(11/9))-3' and has conserved cleavage positions. Both enzymes resemble two other class-IIS endonucleases from Thermus sp.: TaqII and Tth111II. N-terminal amino acid sequences of TspGWI tryptic peptides exhibit 88.9-100% similarity to the TaqII sequence. All four enzymes were purified to homogeneity; their polypeptide sizes (114.5-122 kDa) make them the largest class-IIS restriction endonucleases known to date. The existence of a Thermus sp. sub-family of class-IIS restriction endonucleases of a common origin is herein proposed.     

Cloning and expression of the BalI restriction-modification system.

BalI, a type II restriction-modification (R-M) system from the bacterium, Brevibacterium albidum, recognizes the DNA sequence 5'-TGGCCA-3'. We cloned the genes encoding the BalI restriction endonuclease and methyltransferase and expressed them in Escherichia coli. The two genes were aligned tail-to-tail and their termination codons overlapped. BalI restriction endonuclease and methyltransferase comprise 260 and 280 amino acids, respectively, and have molecular weights of 29 043 and 31 999 Da. The amino acid sequence of BalI methyltransferase is similar to that of other m6A MTases, although it has been categorized as a m5C methyltransferase. A high expression system for the BalI restriction endonuclease was constructed in E. coli for the production of large quantities of enzyme.     

Recognition sequence of restriction endonuclease KpnI from Klebsiella pneumoniae.

We have determined the recognition sequence of the restriction endonuclease KpnI, previously isolated from Klebsiella pneumoniae. The enzyme cleaves the twofold rotationally symmetric sequence (see book for formula) at the positions indicated by the arrows, producing 3' protruding cohesive ends, four nucleotides in length. The specific cleavage site was unambiguously deduced using both 3' and 5' end analyses of KpnI generated restriction fragments of simian-virus 40 (SV40) DNA (1 site), adenovirus-2 (Ad-2) DNA (8 sites), and a plasmid (pCRI) DNA (2 sites).     

Characterization of restriction endonuclease AjoI from Acinetobacter johnsonii

Abstract A new type II restriction endonuclease, named Ajo I, was detected in Acinetobacter johnsonii . The enzyme Ajo I, an isoschizomer of Pst I, recognized the hexanucleotide sequence [5'-CTGCA/G-3'], with a cleavage site generating fragments of DNA with protruding cohesive 3' termini.     

AbeI, a restriction endonuclease from Azotobacter beijerinckii, which recognizes the asymmetric heptanucleotide sequence 5'-CCTCAGC-3'(-5/-2).

A new restriction endonuclease Abe I has beenisolated from Azotobacter beijerinckii. This enzymerecognizes the asymmetric heptanucleotide sequence 5'-CCTCAGC-3' and cleaves within it symmetrically at positions -5/-2 in the opposing strands, producing three base protruding 5'-ends.     

Cloning of the BssHII restriction-modification system in Escherichia coli : BssHII methyltransferase contains circularly permuted cytosine-5 methyltransferase motifs.

BssHII restriction endonuclease cleaves 5'-GCGCGC-3' on double-stranded DNA between the first and second bases to generate a four base 5'overhang. BssHII restriction endonuclease was purified from the native Bacillus stearothermophilus H3 cells and its N-terminal amino acid sequence was determined. Degenerate PCR primers were used to amplify the first 20 codons of the BssHII restriction endonuclease gene. The BssHII restriction endonuclease gene (bssHIIR) and the cognate BssHII methyltransferase gene (bssHIIM) were cloned in Escherichia coli by amplification of Bacillus stearothermophilus genomic DNA using PCR and inverse PCR. BssHII methyltransferase (M.BssHII) contains all 10 conserved cytosine-5 methyltransferase motifs, but motifs IX and X precede motifs I-VIII. Thus, the conserved motifs of M. BssHII are circularly permuted relative to the motif organizations of other cytosine-5 methyltransferases. M.BssHII and the non-cognate multi-specific phiBssHII methyltransferase, M.phiBss HII [Schumann,J. et al . (1995) Gene, 157, 103-104] share 34% identity in amino acid sequences from motifs I-VIII, and 40% identity in motifs IX-X. A conserved arginine is located upstream of a TV dipeptide in the N-terminus of M.BssHII that may be responsible for the recognition of the guanine 5' of the target cytosine. The BssHII restriction endonuclease gene was expressed in E.coli via a T7 expression vector.