Type I restriction enzymes and their relatives

Type I restriction enzymes (REases) are large pentameric proteins with separate restriction (R), methylation (M) and DNA sequence-recognition (S) subunits. They were the first REases to be discovered and purified, but unlike the enormously useful Type II REases, they have yet to find a place in the enzymatic toolbox of molecular biologists. Type I enzymes have been difficult to characterize, but this is changing as genome analysis reveals their genes, and methylome analysis reveals their recognition sequences. Several Type I REases have been studied in detail and what has been learned about them invites greater attention. In this article, we discuss aspects of the biochemistry, biology and regulation of Type I REases, and of the mechanisms that bacteriophages and plasmids have evolved to evade them. Type I REases have a remarkable ability to change sequence specificity by domain shuffling and rearrangements. We summarize the classic experiments and observations that led to this discovery, and we discuss how this ability depends on the modular organizations of the enzymes and of their S subunits. Finally, we describe examples of Type II restriction–modification systems that have features in common with Type I enzymes, with emphasis on the varied Type IIG enzymes.     

Type II restriction endonucleases from Bacillus sphaericus

Abstract The galactophilic lectin of the bacterium Pseudomonas aeruginosa (PA-I) was used for mitogenic stimulation of peripheral bloodlymphocytes from cancer-bearing patients and healthy subjects. This lectin, which preferentially stimulates sialidase-treated lymphocytes, was shown to be useful in the detection of an impairment in the mitogenic response of the patients' lymphocytes. Its efficiency was at least as that of the Phaseolus vulgaris lectin (PHA), which is widely used for the diagnosis and prognosis of deficient immunocompetence states.     

On the DNA cleavage mechanism of Type I restriction enzymes

Although the DNA cleavage mechanism of Type I restriction–modification enzymes has been extensively studied, the mode of cleavage remains elusive. In this work, DNA ends produced by EcoKI, EcoAI and EcoR124I, members of the Type IA, IB and IC families, respectively, have been characterized by cloning and sequencing restriction products from the reactions with a plasmid DNA substrate containing a single recognition site for each enzyme. Here, we show that all three enzymes cut this substrate randomly with no preference for a particular base composition surrounding the cleavage site, producing both 5′- and 3′-overhangs of varying lengths. EcoAI preferentially generated 3′-overhangs of 2–3 nt, whereas EcoKI and EcoR124I displayed some preference for the formation of 5′-overhangs of a length of ~6–7 and 3–5 nt, respectively. A mutant EcoAI endonuclease assembled from wild-type and nuclease-deficient restriction subunits generated a high proportion of nicked circular DNA, whereas the wild-type enzyme catalyzed efficient cleavage of both DNA strands. We conclude that Type I restriction enzymes require two restriction subunits to introduce DNA double-strand breaks, each providing one catalytic center for phosphodiester bond hydrolysis. Possible models for DNA cleavage are discussed.     

Type II restriction endonucleases cleave single-stranded DNAs in general.

Restriction endonucleases (13 out of 18 species used for the test) were certified to cleave single-stranded(ss)DNA. Such enzymes as AvaII, HaeII, DdeI, AluI, Sau3AI, AccII,TthHB8I and HapII were newly reported to cleave ssDNA. A model to account for the cleavage of ssDNA by restriction enzymes was proposed with supportive data. The essential part of the model was that restriction enzymes preferentially cleave transiently formed secondary structures (called canonical structures) in ssDNA composed of two recognition sequences with two fold rotational symmetry. This means that a restriction enzyme can cleave ssDNAs in general so far as the DNAs have the sequences of restriction sites for the enzyme, and that the rate of cleavage depends on the stabilities of canonical structures.     

MnlI--The member of H-N-H subtype of Type IIS restriction endonucleases

The Type IIS restriction endonuclease MnlI recognizes the non-palindromic nucleotide sequence 5'-CCTC(N)7/6 downward arrow and cleaves DNA strands as indicated by the arrow. The genes encoding MnlI restriction-modification system were cloned and sequenced. It comprises N6-methyladenine and C5-methylcytosine methyltransferases and the restriction endonuclease. Biochemical studies revealed that MnlI restriction endonuclease cleaves double- and single-stranded DNA, and that it prefers different metal ions for hydrolysis of these substrates. Mg2+ ions were shown to be required for the specific cleavage of double-stranded DNA, whereas Ni2+ and some other transition metal ions were preferred for nonspecific cleavage of single-stranded DNA. The C-terminal part of MnlI restriction endonuclease revealed an intriguing similarity with the H-N-H type nucleolytic domain of bacterial toxins, Colicin E7 and Colicin E9. Alanine replacements in the conserved sequence motif 306Rx3ExHHx14Nx8H greatly reduced specific activity of MnlI, and some mutations even completely inactivated the enzyme. However, none of these mutations had effect on MnlI binding to the specific DNA, and on its oligomerisation state as well. We interpret the presented experimental evidence as a suggestion that the motif 306Rx3ExHHx14Nx8H represents the active site of MnlI. Consequentially, MnlI seems to be the member of Type IIS with the active site of the H-N-H type.     

A general assay for restriction endonucleases and other DNA-modifying enzymes with plasmid substrates

A procedure for measuring the activities of enzymes that alter the covalent structure of DNA is described. The assay utilizes covalently closed circles of DNA as the substrate and yields quantitative data on the fraction of this DNA converted to both open-circle and linear forms.     

Site-specific restriction endonucleases in cyanobacteria

AIM: Planktic cyanobacteria were screened for endodeoxyribonucleases. Principal component analysis (PCA) was employed to demonstrate a potential relationship between certain enzymes and a group of cyanobacteria. The data were obtained from a data bank and this study. METHODS AND RESULTS: Enzymes were partially purified using column chromatography. Anabaena strains contained Asp83/1I (5'-TTCGAA-3'), Asp83/1II (5'-GGCC-3'), Asp90I (5'-ACRYGT-3') and five isoschizomeric enzymes (5'-ATCGAT-3'). Aphanizomenon and Microcystis strains contained ApcTR183I (5'-TGCGCA-3') and Msp199I (5'-CCGG-3'), respectively. Planktothrix strains possessed Psc2I (5'-GAANNNNTTC-3'), Psc27I and Psc28I (5'-TTCGAA-3'). PCA showed that the most common cyanobacterial endonuclease types were AvaII, AvaI and AsuII. CONCLUSIONS: All planktic cyanobacteria studied contained restriction endonucleases. The defined restriction endonucleases were isoschizomers of known enzymes. The Nostoc and the Spirulina genera had an association, while the majority of the genera had no association with certain endonuclease type(s). SIGNIFICANCE AND IMPACT OF THE STUDY: The defined enzymes in this study and the estimated trend in the endonuclease type distribution allow more efficient avoidance of cyanobacterial restriction barriers.     

A nomenclature for restriction enzymes,DNA methyltransferases,homing endonucleases and their genes

A nomenclature is described for restriction endonucleases, DNA methyltransferases, homing endonucleases and related genes and gene products. It provides explicit categories for the many different Type II enzymes now identified and provides a system for naming the putative genes found by sequence analysis of microbial genomes.     

Screening for catalytically active Type II restriction endonucleases using segregation-induced methylation deficiency

Type II restriction endonucleases (REases) are one of the basic tools of recombinant DNA technology. They also serve as models for elucidation of mechanisms for both site-specific DNA recognition and cleavage by proteins. However, isolation of catalytically active mutants from their libraries is challenging due to the toxicity of REases in the absence of protecting methylation, and techniques explored so far had limited success. Here, we present an improved SOS induction-based approach for in vivo screening of active REases, which we used to isolate a set of active variants of the catalytic mutant, Cfr10I^E204Q. Detailed characterization of plasmids from 64 colonies screened from the library of ∼200 000 transformants revealed 29 variants of cfr10IR gene at the level of nucleotide sequence and 15 variants at the level of amino acid sequence, all of which were able to induce SOS response. Specific activity measurements of affinity-purified mutants revealed >200-fold variance among them, ranging from 100% (wild-type isolates) to 0.5% (S188C mutant), suggesting that the technique is equally suited for screening of mutants possessing high or low activity and confirming that it may be applied for identification of residues playing a role in catalysis.     

Type II restriction endonucleases: structural, functional and evolutionary relationships.

Type II restriction endonucleases are a paradigm for site-specific cleavage of DNA. Recent structural analyses, in particular in the presence of various divalent metals, have shed new insight into the mechanisms of catalysis. In addition, during this past year the crystal structure determinations of MutH, lambda-exonuclease and FokI have revealed that these proteins are also members of the same family.     

Genomic analysis of phase I and II Coxiella burnetii with restriction endonucleases

Restriction endonuclease-digested DNAs from several isolates of phase I and phase II Coxiella burnetii were compared using agarose gel electrophoresis and soft-laser scanning densitometry. Our results demonstrate that the two phases are, as previously assumed, alternative phases of the same organism. Although the restriction endonuclease digestion revealed genetic differences between clonal isolates of phase I and phase II C. burnetii Nine Mile strain, these differences do not appear to be related to antigenic phase variation. However, analyses of the fragment patterns generated by restriction enzyme digestion suggest potential grouping of the different isolates.     

Mesophilic cyanobacteria producing thermophilic restriction endonucleases

When searching for the site-specific endonucleases in several strains of Phormidium we made the following observations. Among the 16 strains that originated from 15 species of Phormidium, 12 produced one or more restriction enzymes, of which two produced the highly thermophilic restriction endonucleases PtaI and PpaAII with their optimum activity at 65-80 degrees C, which is far above the lethal temperature for the host microorganism (40 degrees C). These two temperature-resistant enzymes are isoschizomers of known BspMII and TaqI endonucleases, respectively. The presence of the thermophilic TaqI isoschizomer does not seem to play any role in the mesophilic host microorganism, which does not even contain an active cognate methyltransferase. Among the remaining 10 strains, six produced isoschizomers of endonucleases which we first described in cyanobacteria, namely: PfaAII (NdeI), PinBII and PtaI (BspMII), PlaAII (RsalI), PpaAII, PpeI (ApaI). Two enzymes, PauAII (AhaIII) and PfaAII (NdeI), belong to a group of a very rarely occurring isoschizomers. Out of 21 cyanobacterial endonucleases investigated by us, four were active in a wide range of temperatures (from 15 to 60 degrees C) which also extended the optimal growth temperature of the hosts. We assume that our observation on the presence of temperature-resistant restriction enzymes in mesophilic hosts supports the idea of horizontal gene transfer. Restriction modification systems may be an excellent tool for investigation of that phenomenon.     

Solitary restriction endonucleases in prokaryotic genomes

Prokaryotic restriction-modification (R-M) systems defend the host cell from the invasion of a foreign DNA. They comprise two enzymatic activities: specific DNA cleavage activity and DNA methylation activity preventing cleavage. Typically, these activities are provided by two separate enzymes: a DNA methyltransferase (MTase) and a restriction endonuclease (RE). In the absence of a corresponding MTase, an RE of Type II R-M system is highly toxic for the cell. Genes of the R-M system are linked in the genome in the vast majority of annotated cases. There are only a few reported cases in which the genes of MTase and RE from one R-M system are not linked. Nevertheless, a few hundreds solitary RE genes are present in the Restriction Enzyme Database (http://rebase.neb.com) annotations. Using the comparative genomic approach, we analysed 272 solitary RE genes. For 57 solitary RE genes we predicted corresponding MTase genes located distantly in a genome. Of the 272 solitary RE genes, 99 are likely to be fragments of RE genes. Various explanations for the existence of the remaining 116 solitary RE genes are also discussed.     

Site-specific restriction endonucleases in Bacillus licheniformis

Abstract We systematically studied site-specific restriction endonucleases in Bacillus licheniformis strains and detected endonuclease activity in 25 of 217 strains tested. Three different activities were obtained. One of these activities detected in 21 strains was the most representative within the species and produced a banding pattern, after digestion of A DNA, identical to that seen with Cla I. Two other strains isolated from soil samples from China and USA were found to produce a DNA-cleaving enzyme with the same recognition sequence as Bsa I. One producer strain, isolated from a Peruvian soil sample, showed to possess a mixture of two isoschizomers, Cla I and Bsa I. Finally, one strain produced an endonuclease activity, not previously described in B. licheniformis , that showed the same recognition sites as Bsu 361.     

Chromosomal aberrations induced by restriction endonucleases

Restriction endonucleases (REs) are able to induce chromosomal aberrations in Chinese hamster ovary (CHO) cells. The G1 phase of the cell cycle seems to be especially sensitive for the induction of chromosomal aberrations by REs. The different capacities of REs to induce chromosomal aberrations are probably correlated with the number of recognition sites in the genome.