Synthesis of decadeoxyribonucleotides containing 5-modified uracils and their interactions with restriction endonucleases Bgl II, Sau 3AI and Mbo I (nucleosides and nucleotides 82)

Decadeoxyribonucleotides containing uracil, 5-bromouracil, 5-cyanouracil and 5-ethyluracil in recognition sequences of restriction endonucleases Bgl II, Sau 3AI, Mbo I were synthesized. Decanucleotides containing 5-bromouracil in place of thymine had essentially the same susceptibility to all the restriction endonucleases. Uracil-containing decanucleotides were however very resistant to attack. Decanucleotides containing 5-cyanouracil in the recognition sequence were strongly resistant to hydrolysis by Sau 3AI, but were hydrolysed by Bgl II and Mbo I as well as the parent decanucleotide. Decanucleotides containing 5-ethyluracil were strongly resistant to hydrolysis by Sau 3AI, but were partially resistant to hydrolysis by Bgl II and Mbo I.     

Synthesis of deoxyoligonucleotides containing 7-deazaadenine: recognition and cleavage by restriction endonuclease Bgl II and Sau 3AI (nucleosides and nucleotides Part 55).

Deoxydecanucleotides having a recognition sequence of Bgl II and Sau 3AI, and their 7-deazaadenine analogs were synthesized. The decanucleotides containing 7-deazaadenine in place of adenine were partially resistant to the hydrolysis by Sau 3AI and strongly resistant to that by Bgl II. A new hypothesis on the mode of recognition and cleavage of specific nucleotide sequences by Bgl II, recognizing one strand and cleaving the other strand, is presented.     

Synthesis of decadeoxyribonucleotides containing N6-methyladenine, N4-methylcytosine, and 5-methylcytosine: recognition and cleavage by restriction endonucleases (nucleosides and nucleotides part 74).

The naturally-occurring modified bases, N6-methyladenine, N4-methylcytosine, and 5-methylcytosine were chemically introduced in place of the adenine or cytosine in the decadeoxyribonucleotides containing recognition sequences of Bgl II, Sau 3AI, Mbo I and Mfl I. The modified oligomers bind to the enzymes but the rates of cleavage by the enzymes are variable.     

Oligodeoxynucleotides containing 7-deazaadenine: synthesis and recognition by restriction endonucleases

Deoxydecanucleotides containing a recognition sequence of Bg1 II and Sau 3AI, and their 7-deazaadenine analogs were synthesized by the phosphotriester method. The decanucleotides containing 7-deazaadenine in place of adenine were partially or strongly resistant to the hydrolysis by these restriction endonucleases.     

The interface between an alternating CG motif and a random sequence motif displays altered nuclease activity.

Previously we described the B-Z junctions produced in oligomers containing (5meCG)4 segments in the presence of 5.0 M NaCl or 50 uM Co(NH3)6+3 [Sheardy, R.D. & Winkle, S.A., Biochemistry 28, 720-725 (1989); Winkle, S.A., Aloyo, M.C., Morales, N., Zambrano, T.Y. & Sheardy, R.D., Biochemistry 30, 10601-10606 (1991)]. The circular dichroism spectra of an analogous unmethylated oligomer containing (CG)4, termed BZ-IV, in 5.0 M NaCl and in 50 uM Co(NH3)+3 suggest, however, that this oligomer does not form a B-Z hybrid. BZ-IV possesses Hha I sites (CGCG) in the (CG)4 segment and an Mbo I site (GATC) at the terminus of the (CG)4 segment. BZ-IV is equally digestible in the presence and absence of cobalt hexamine by Hha I, further indicating that the structure of BZ-IV is fully B-like under these conditions. The Mbo I cleavage site at the juncture between the (CG)4 segment and the adjacent random segment displays enhanced cleavage by both Mbo I and its isoschizomer Sau3AI in the presence of cobalt hexamine. In addition, exonuclease III digestion of BZ-IV is inhibited at this juncture. Actinomycin inhibits Mbo I activity in the presence of cobalt hexamine but not in the absence. Together, these results suggest that enzymes recognize the interfaces of (CG)n and adjacent random sequences as altered substrates even in the absence of a B-Z junction formation.     

Sau3AI, a monomeric type II restriction endonuclease that dimerizes on the DNA and thereby induces DNA loops

Here, we report that Sau3AI, an unusually large type II restriction enzyme with sequence homology to the mismatch repair protein MutH, is a monomeric enzyme as shown by gel filtration and ultracentrifugation. Structural similarities in the N- and C-terminal halves of the protein suggest that Sau3AI is a pseudo-dimer, i.e. a polypeptide with two similar domains. Since Sau3AI displays a nonlinear dependence of cleavage activity on enzyme concentration and a strong preference for substrates with two recognition sites over those with only one, it is likely that the functionally active form of Sau3AI is a dimer of a pseudo-dimer. Indeed, electron microscopy studies demonstrate that two distant recognition sites are brought together through DNA looping induced by the simultaneous binding of two Sau3AI molecules to the DNA. We suggest that the dimeric form of Sau3AI supplies two DNA-binding sites, one that is associated with the catalytic center and one that serves as an effector site.     

Crystal structure and function of C-terminal Sau3AI domain

Sau3AI is a type II restriction enzyme that recognizes the 5'-GATC-3' sequence in double-strand DNA and cleaves at 5' to the G residue. The C-terminal domain of Sau3AI (Sau3AI-C), which contains amino acids from 233 to 489, was crystallized and its structure was solved by using the Multi-wavelength Anomalous Diffraction method. The Sau3AI-C structure at 1.9 A resolution is similar to the structure of MutH, a DNA mismatch repair protein that shares high sequence similarity with the N-terminal Sau3AI domain. The functional analysis shows that Sau3AI-C can bind DNA with one recognition sequence but has no cleavage activity. These results indicate that Sau3AI is a pseudo-dimer belonging to the type IIe restriction enzymes and the Sau3AI-C is the allosteric effector domain that assists DNA binding and cleavage.     

Characterization of a novel type II restriction-modification system, Sth368I, encoded by the integrative element ICESt1 of Streptococcus thermophilus CNRZ368

A novel type II restriction and modification (R-M) system, Sth368I, which confers resistance to phiST84, was found in Streptococcus thermophilus CNRZ368 but not in the very closely related strain A054. Partial sequencing of the integrative conjugative element ICESt1, carried by S. thermophilus CNRZ368 but not by A054, revealed a divergent cluster of two genes, sth368IR and sth368IM. The protein sequence encoded by sth368IR is related to the type II endonucleases R.LlaKR2I and R.Sau3AI, which recognize and cleave the sequence 5'-GATC-3'. The protein sequence encoded by sth368IM is very similar to numerous type II 5-methylcytosine methyltransferases, including M.LlaKR2I and M.Sau3AI. Cell extracts of CNRZ368 but not A054 were found to cleave at the GATC site. Furthermore, the C residue of the sequence 5'-GATC-3' was found to be methylated in CNRZ368 but not in A054. Cloning and integration of a copy of sth368IR and sth368IM in the A054 chromosome confers on this strain phenotypes similar to those of CNRZ368, i.e., phage resistance, endonuclease activity of cell extracts, and methylation of the sequence 5'-GATC-3'. Disruption of sth368IR removes resistance and restriction activity. We conclude that ICESt1 encodes an R-M system, Sth368I, which recognizes the sequence 5'-GATC-3' and is related to the Sau3AI and LlaKR2I restriction systems.     

A model of structure and action of Sau3AI restriction endonuclease that comprises two MutH-like endonuclease domains within a single polypeptide

Sau3AI is a type II endonuclease that cleaves GATC sequences, producing sticky ends with 4-nucleotide 5'-overhangs. Its activity is inhibited by cytosine C5-methylation within the target sequence. In the N-terminus, Sau3AI exhibits sequence similarity to the GATC-specific single-strand nicking endonuclease MutH implicated in mismatch repair (Ban and Yang, 1998). Sequence analysis of Sau3AI and its homologs reveals that Sau3AI possesses an additional MutH-like domain in the C-terminus. Structure prediction suggests that the C-terminal domain lacks the endonuclease active site but retains all putative DNA-binding elements. As an illustration of these findings, a model of quaternary structure of Sau3AI complexed with the target DNA is presented. These predictions have implications for evolution, structure and function of bacterial DNA repair enzymes and restriction endonucleases.     

Cytokinins do not noticeably change the methylation of adenine residues of DNA in cultured Escherichia coli B

6-Benzylaminopurine (6-BAP) (1 mg/ml) does not influence the growth of E. coli B cell cultures or the number of [8-14C] labeled N6-methyladenine (m6A) residues in the total DNA [(100.m6A/(A x m6A) = 1.7]. The growth of bacterial cells in the presence of adenine or cytokinins (6-BAP, kinetin, zeatin) (1 mg/ml) was unaccompanied by significant changes in the intracellular content of plasmid pBR 322. The mode of restriction by endonuclease Cfu I hydrolyzing the Gm6ATC site of plasmids pBR 322 from E. coli B cells grown in the presence of adenine or one of the above-mentioned cytokinins is identical. These plasmids also have identical restriction products Mbo I or Sau 3AI. Thus, the cytokinins under study do not markedly affect the methylation of adenine residues in total DNA of E. coli B cell cultures and the GATC sequence in plasmids pBR 322 isolated from these cells.     

Tandem repeats within the inverted terminal repetition of vaccinia virus DNA

A tandemly repeated sequence within the genome of vaccinia virus is cut to fragments of approximately 70 bp by Hinf I, Taq I or Mbo II. The 70 bp repetition was localized within the much larger (10,300 bp) inverted terminal repetition by restriction analysis of cloned DNA fragments and by hybridization of the purified 70 bp repeat to vaccinia virus DNA restriction fragments. The molar abundance of the 70 bp fragment corresponds to a 30 fold repetition at each end of the genome. The repeating restriction endonuclease sites were mapped by agarose gel electrophoresis of partial Hinf I digests of the terminally labeled cloned DNA fragment. The first of 13 repetitive Hinf I sites occurred approximately 150 bp from the end of the cloned DNA. After an intervening sequence of approximately 435 bp, a second series of 17 repetitive Hinf I sites occurred. The DNA between the two blocks of repetitions has a unique sequence containing single Dde I, Alu I and Sau 3A sites. Tandem repeats within the inverted terminal repetition could serve to accelerate self-annealing of single strands of DNA to form circular structures during replication.     

Type II restriction modification systems of Prevotella bryantii TC1-1 and Prevotella ruminicola 23 strains and their effect on the efficiency of DNA introduction via electroporation

The restriction endonucleases PbrTI and Pru2I, isoschizomers of Sau3AI and HaeIII, were partially purified and characterized from anaerobic rumen bacteria Prevotella bryantii TC1-1 and Prevotella ruminicola 23, respectively. These are the first type II restriction endonucleases discovered in strains of the genus Prevotella, and they represent one of the barriers hindering gene transfer in these microorganisms. Heterologous DNA was protected against the action of the PbrTI or Pru2I by incubation in a cell-free extract of the respective strain which contained 20 mM EDTA. This led to the development of a protocol enabling successful electrotransformation of the P. bryantii TC1-1 strain with a pRH3 Bacteroides--Escherichia coli shuttle vector containing up to 7-kb long DNA inserts. Plasmid DNA isolated from the transformed strain facilitated the transfer with further increased efficiency and made possible the introduction of ligation reaction products directly to P. bryantii TC1-1 without passing them first through E. coli.     

Differential digestion of the centromeric heterochromatic regions of the 5-azacytidine-decondensed human chromosomes 1, 9, 15, and 16 by NdeII and Sau3AI restriction endonucleases

A study on the factors involved in chromosome digestion by restriction endonuclease was carried out on 5-azacytidine treated and untreated human chromosomes 1, 9, 15 and 16 by using NdeII and Sau3AI isoschizomers. After treatment with 5-azacytidine, chromosomes 1, 9, 15, and 16 showed two differentiated areas at the centromeric regions: the centromere, fully condensed, and the pericentromeric heterochromatin, decondensed. Chromosomes not treated with 5-azacytidine after digestion with Sau3AI and NdeII showed all the centromeric regions undigested, except pair number 1, digested at the pericentromeric area. Digestion of the 5-azacytidine decondensed chromosomes with Sau3AI and NdeII showed the centromeres undigested in the four chromosome pairs while the pericentromeric heterochromatin appeared largely digested. Other factors, different to target distribution, are necessary to explain the pattern of restriction endonuclease digestion observed in this communication.     

Crystallization and preliminary X-ray analysis of Sau3AI/E64A mutant protein

Sau3AI is a type II restriction endonuclease that recognizes the palindromic sequence 5'-GATC-3' and cleaves 5' to G residue on each strand. The E64A mutant full length protein was cloned and expressed in Escherichia coli. The purified (His) (6)-tagged protein has monomer and dimer fraction and was crystallized by the hanging-drop vapor-diffusion technique. The dimer protein crystals can diffract to 3.0A. resolution and the monomer protein crystals can diffract to better than 2.8A. resolution. One completed dataset has been collected and it shows that the monomer orthorhombic Sau3AI/E64A crystal is in space group C2221 with unit cell parameters (69.44, 197.60, 191.46, 90, 90, 90) and contains two molecules in one asymmetric unit.     

A possible structure for calf satellite DNA I.

Calf satellite DNA I (p = 1.715) has been hydrolysed by a number or restriction endonucleases. It consists of a repeating unit of 1460 nucleotide pairs within which the sites of Eco R II Mbo I, Sac I, Alu I, Ava II and Hha I were localised in comparison with those of Eco R I and Hind II. The distribution of the Hpa II, Sac I, Hha I, Hinf I and Mbo II sites within calf satellite DNA I, as well as that of several restriction endonuclease sites within calf satellite DNA III (p = 1.705) allowed me to define subsatellite fractions. Furthermore, some of the sites of the CpG containing restriction enzymes Hpa II and Hha I are lacking. The possible implications of these results are discussed.     

Host-mediated modification of Sau3AI restriction in Listeria monocytogenes: prevalence in epidemic-associated strains.

Most major food-related outbreaks of listeriosis have been traced to a cluster of genetically related strains of serovar 4b (epidemic clone). In spite of numerous searches, distinct bacteriologic or virulence-related features unique to these strains have eluded identification, although a restriction fragment length polymorphism (RFLP) characteristic of the epidemic clone has previously been described (W. Zheng and S. Kathariou, Appl. Environ. Microbiol. 61:4310-4314, 1995). We found that DNAs from 75 strains which were derived from three separate outbreaks and which had the epidemic clone-specific RFLP were also invariably resistant to digestion by Sau3AI and other restriction endonucleases sensitive to cytosine methylation at 5' GATC 3' sites. This modification of Sau3AI restriction was host mediated, as it did not persist when DNA was cloned and propagated in Escherichia coli, and was uncommon among other Listeria strains. Epidemic-associated strains with this modification were resistant to infection by phage propagated in a serotype 4b strain which was not known to be involved in an epidemic and which lacked the epidemic clone-specific RFLP. Screening for susceptibility to MboI digestion revealed that these epidemic strains lacked methylation of adenines at GATC sites. This type of modification was rare among Listeria strains and was found in only three (of eight screened) strains of serovar 1/2b, possibly representing one clonal lineage.     

Genetic variation in native and introduced populations of the 'New Zealand flatworm', Arthurdendyus triangulatus

The internal transcribed spacer regions (ITS1 and ITS2) including the 5.8S region of the ‘New Zealand flatworm’, Arthurdendyus triangulates, are 1004 base pairs in length. Restriction fragment length polymorphism analysis of PCR products (PCR‐RFLP) was conducted on A. triangulates specimens from 45 locations in Northern Ireland, Scotland, England and New Zealand. Seven restriction endonucleases (Alu I, Rsa I, Sau3A I, Cfo I, Nde I, Dde I, and Mbo I) were used to reveal intraspecific variation. Analysis of molecular variance revealed the presence of population genetic substructuring, with most genetic heterogeneity present between populations rather than between individuals or geographical regions. No distinct differences were found between Northern Irish and Scottish populations but phylogenetic analysis supports the hypothesis of multiple introductions from New Zealand. There was no significant relationship between genetic distance and geographic distance, as would be expected for natural spread, indicating that this species is largely anthropochorous, even in parts of New Zealand.     

Single-strand and double-strand cleavage at half-modified and fully modified recognition sites for the restriction nucleases Sau3a and Taqi

The influence of cytosine methylation on the cleavage of DNA by the restriction nucleases Sau3A and TaqI has been investigated. Bovine satellite DNA fragments containing a GATCGA sequence, i.e. a Sau3A site overlapping with a TaqI site have been used in this study. The methylation of these fragments has been determined by sequence analysis. It has been found that a TaqI site (TCGA) methylated at cytosine in both DNA strands is still sensitive to double-strand cleavage. A Sau3A site (GATC), however, is rendered resistant to double-strand cleavage by methylation of a single cytosine. Fragments containing the "half-modified" Sau3A site are nicked in the unmethylated DNA strand. It has been shown by sequence analysis of nicked DNA that the single-strand break occurs at the same position which is cleaved in unmodified DNA.