Type I restriction endonucleases are true catalytic enzymes

Type I restriction endonucleases are intriguing, multifunctional complexes that restrict DNA randomly, at sites distant from the target sequence. Restriction at distant sites is facilitated by ATP hydrolysis-dependent, translocation of double-stranded DNA towards the stationary enzyme bound at the recognition sequence. Following restriction, the enzymes are thought to remain associated with the DNA at the target site, hydrolyzing copious amounts of ATP. As a result, for the past 35 years type I restriction endonucleases could only be loosely classified as enzymes since they functioned stoichiometrically relative to DNA. To further understand enzyme mechanism, a detailed analysis of DNA cleavage by the EcoR124I holoenzyme was done. We demonstrate for the first time that type I restriction endonucleases are not stoichiometric but are instead catalytic with respect to DNA. Further, the mechanism involves formation of a dimer of holoenzymes, with each monomer bound to a target sequence and, following cleavage, each dissociates in an intact form to bind and restrict subsequent DNA molecules. Therefore, type I restriction endonucleases, like their type II counterparts, are true enzymes. The conclusion that type I restriction enzymes are catalytic relative to DNA has important implications for the in vivo function of these previously enigmatic enzymes.     

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.     

Comparative characterization of the PvuRts1I family of restriction enzymes and their application in mapping genomic 5-hydroxymethylcytosine

PvuRts1I is a modification-dependent restriction endonuclease that recognizes 5-hydroxymethylcytosine (5hmC) as well as 5-glucosylhydroxymethylcytosine (5ghmC) in double-stranded DNA. Using PvuRts1I as the founding member, we define a family of homologous proteins with similar DNA modification-dependent recognition properties. At the sequence level, these proteins share a few uniquely conserved features. We show that these enzymes introduce a double-stranded cleavage at the 3'-side away from the recognized modified cytosine. The distances between the cleavage sites and the modified cytosine are fixed within a narrow range, with the majority being 11-13 nt away in the top strand and 9-10 nt away in the bottom strand. The recognition sites of these enzymes generally require two cytosines on opposite strand around the cleavage sites, i.e. 5'-CN(11-13)↓N(9-10)G-3'/3'-GN(9-10)↓N(11-13)C-5', with at least one cytosine being modified for efficient cleavage. As one potential application for these enzymes is to provide useful tools for selectively mapping 5hmC sites, we have compared the relative selectivity of a few PvuRts1I family members towards different forms of modified cytosines. Our results show that the inherently different relative selectivity towards modified cytosines can have practical implications for their application. By using AbaSDFI, a PvuRts1I homolog with the highest relative selectivity towards 5ghmC, to analyze rat brain DNA, we show it is feasible to map genomic 5hmC sites close to base resolution. Our study offers unique tools for determining more accurate hydroxymethylomes in mammalian cells.     

S-Adenosyl-L-methionine-dependent restriction enzymes

Restriction-modification (R-M) enzymes are classified into type I, II, III, and IV, based on their recognition sequence, subunit composition, cleavage position, and cofactor requirements. While the role of S-Adenosyl-L-methionine (AdoMet) as the methyl group donor in the methylation reaction is undisputed, its requirement in DNA cleavage reaction has been subject to intense study. AdoMet is a prerequisite for the DNA cleavage by most type I enzymes known so far, with the exception of R.EcoR124I. A number of new type II restriction enzymes belonging to the type IIB and IIG family were found to show AdoMet dependence for their cleavage reaction. The type III enzymes have been found to require AdoMet for their restriction function. AdoMet functions as an allosteric effector of the DNA cleavage reaction and has been shown to bring about conformational changes in the protein upon binding.     

On the structure and operation of type I DNA restriction enzymes.

Type I DNA restriction enzymes are large, molecular machines possessing DNA methyltransferase, ATPase, DNA translocase and endonuclease activities. The ATPase, DNA translocase and endonuclease activities are specified by the restriction (R) subunit of the enzyme. We demonstrate that the R subunit of the Eco KI type I restriction enzyme comprises several different functional domains. An N-terminal domain contains an amino acid motif identical with that forming the catalytic site in simple restriction endonucleases, and changes within this motif lead to a loss of nuclease activity and abolish the restriction reaction. The central part of the R subunit contains amino acid sequences characteristic of DNA helicases. We demonstrate, using limited proteolysis of this subunit, that the helicase motifs are contained in two domains. Secondary structure prediction of these domains suggests a structure that is the same as the catalytic domains of DNA helicases of known structure. The C-terminal region of the R subunit can be removed by elastase treatment leaving a large fragment, stable in the presence of ATP, which can no longer bind to the other subunits of Eco KI suggesting that this domain is required for protein assembly. Considering these results and previous models of the methyltransferase part of these enzymes, a structural and operational model of a type I DNA restriction enzyme is presented.     

Evidence for a repeating domain in type I restriction enzymes.

The primary structures of the recognition subunit (hsdS) in type I restriction enzymes from three isolates of Escherichia coli were compared and aligned by use of amino acid physical properties. A repeating domain was found in each of the subunits suggesting a pseudo-dimeric structure. Secondary structure predictions delineated two helical regions in each domain which suggested that the recognition subunits may act in a fashion similar to that proposed for repressor and activator molecules; namely, interaction with double-stranded DNA through helices and in two successive major grooves on the same DNA side. One helical motif could provide the specific recognition site and the other, the restriction site.     

Nucleoside triphosphate-dependent restriction enzymes

The known nucleoside triphosphate-dependent restriction enzymes are hetero-oligomeric proteins that behave as molecular machines in response to their target sequences. They translocate DNA in a process dependent on the hydrolysis of a nucleoside triphosphate. For the ATP-dependent type I and type III restriction and modification systems, the collision of translocating complexes triggers hydrolysis of phosphodiester bonds in unmodified DNA to generate double-strand breaks. Type I endonucleases break the DNA at unspecified sequences remote from the target sequence, type III endonucleases at a fixed position close to the target sequence. Type I and type III restriction and modification (R-M) systems are notable for effective post-translational control of their endonuclease activity. For some type I enzymes, this control is mediated by proteolytic degradation of that subunit of the complex which is essential for DNA translocation and breakage. This control, lacking in the well-studied type II R-M systems, provides extraordinarily effective protection of resident DNA should it acquire unmodified target sequences. The only well-documented GTP-dependent restriction enzyme, McrBC, requires methylated target sequences for the initiation of phosphodiester bond cleavage.     

Four new type I restriction enzymes identified in Escherichia coli clinical isolates

Using a plasmid transformation method and the RM search computer program, four type I restriction enzymes with new recognition sites and two isoschizomers (EcoBI and Eco377I) were identified in a collection of clinical Escherichia coli isolates. These new enzymes were designated Eco394I, Eco826I, Eco851I and Eco912I. Their recognition sequences were determined to be GAC(5N)RTAAY, GCA(6N)CTGA, GTCA(6N)TGAY and CAC(5N)TGGC, respectively. A methylation sensitivity assay, using various synthetic oligonucleotides, was used to identify the adenines that prevent cleavage when methylated (underlined). These results suggest that type I enzymes are abundant in E.coli and many other bacteria, as has been inferred from bacterial genome sequencing projects.     

REBASE: restriction enzymes and methyltransferases

REBASE contains comprehensive information about restriction enzymes, DNA methyltransferases and related proteins such as nicking enzymes, specificity subunits and control proteins. It contains published and unpublished references, recognition and cleavage sites, isoschizomers, commercial availability, crystal and sequence data. Homing endonucleases are also included. REBASE contains the most complete and up-to-date information about the methylation sensitivity of restriction endonucleases. In addition, there is extensive information about the known and putative restriction-modification (R-M) systems in more than 100 sequenced bacterial and archaeal genomes. The data is available on the web (http://rebase.neb.com/rebase/rebase.html), through ftp (ftp.neb.com) and as monthly updates via email.     

REBASE - restriction enzymes and methylases

REBASE is a comprehensive database of information about restriction enzymes and related proteins. It contains published and unpublished references, recognition and cleavage sites, isoschizomers, commercial availability, methylation sensitivity, crystal and sequence data. DNA methyltransferases, homing endonucleases, nicking enzymes, specificity subunits and control proteins are also included. Most recently, putative DNA methyltransferases and restriction enzymes, as predicted from analysis of genomic sequences, are also listed. The data is distributed via Email, ftp (ftp.neb.com), and the Web (http://rebase.neb.com).     

Real-time observation of DNA looping dynamics of Type IIE restriction enzymes NaeI and NarI

Many restriction enzymes require binding of two copies of a recognition sequence for DNA cleavage, thereby introducing a loop in the DNA. We investigated looping dynamics of Type IIE restriction enzymes NaeI and NarI by tracking the Brownian motion of single tethered DNA molecules. DNA containing two endonuclease recognition sites spaced a few 100 bp apart connect small polystyrene beads to a glass surface. The position of a bead is tracked through video microscopy. Protein-mediated looping and unlooping is then observed as a sudden specific change in Brownian motion of the bead. With this method we are able to directly follow DNA looping kinetics of single protein–DNA complexes to obtain loop stability and loop formation times. We show that, in the absence of divalent cations, NaeI induces DNA loops of specific size. In contrast, under these conditions NarI mainly creates non-specific loops, resulting in effective DNA compaction for higher enzyme concentrations. Addition of Ca²⁺ increases the NaeI-DNA loop lifetime by two orders of magnitude and stimulates specific binding by NarI. Finally, for both enzymes we observe exponentially distributed loop formation times, indicating that looping is dominated by (re)binding the second recognition site.     

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.     

Two type I restriction enzymes from Salmonella species. Purification and DNA recognition sequences

We have purified the type I restriction enzymes SB and SP from Salmonella typhimurium and S. potsdam, respectively, and determined the DNA sequences that they recognize. These sequences resemble those previously determined for the type I enzymes, EcoB, EcoK and EcoA, in that the specific part of the sequence is divided into two domains by a spacer of non-specific sequence that has a fixed length for each enzyme. Two main differences from the previously determined sequences are seen. Both of the new sequences are degenerate and one of them, SB, has one trinucleotide and one pentanucleotide-specific domain rather than the trinucleotide and tetranucleotide domains seen for all of the other enzymes. The only conserved features of the recognition sequences are the adenosyl residues that are methylated in the modification reaction. For all of the enzymes these are situated ten or 11 base-pairs apart, one on each strand of the DNA. This suggests that the enzymes bind to DNA along one face of the double helix making protein-DNA interaction in two successive major grooves with most of the non-specific spacer sequence in the intervening minor groove.     

A RecB-family nuclease motif in the Type I restriction endonuclease EcoR124I

The Type I restriction-modification enzyme EcoR124I is an ATP-dependent endonuclease that uses dsDNA translocation to locate and cleave distant non-specific DNA sites. Bioinformatic analysis of the HsdR subunits of EcoR124I and related Type I enzymes showed that in addition to the principal PD-(E/D)xK Motifs, I, II and III, a QxxxY motif is also present that is characteristic of RecB-family nucleases. The QxxxY motif resides immediately C-terminal to Motif III within a region of predicted alpha-helix. Using mutagenesis, we examined the role of the Q and Y residues in DNA binding, translocation and cleavage. Roles for the QxxxY motif in coordinating the catalytic residues or in stabilizing the nuclease domain on the DNA are discussed.     

Type I restriction enzyme with RecA protein promotes illegitimate recombination

Illegitimate (non-homologous) recombination requires little or no sequence homology between recombining DNAs and has been regarded as being a process distinct from homologous recombination, which requires a long stretch of homology between recombining DNAs. However, we have found a type of illegitimate recombination that requires an interaction between long homologous DNA sequences. It was detected when a plasmid that carried 2-kb-long inverted repeats was subjected to type I (EcoKI) restriction in vivo within a special mutant strain of Escherichia coli. In the present work, we analyzed genetic requirements for this type of illegitimate recombination in well-defined genetic backgrounds. Our analysis demonstrated dependence on RecA function and on the presence of two EcoKI sites on the substrate DNA. These results are in harmony with a model in which EcoKI restriction enzyme attacks an intermediate of homologous recombination to divert it to illegitimate recombination.     

Structural and evolutionary classification of Type II restriction enzymes based on theoretical and experimental analyses

For a very long time, Type II restriction enzymes (REases) have been a paradigm of ORFans: proteins with no detectable similarity to each other and to any other protein in the database, despite common cellular and biochemical function. Crystallographic analyses published until January 2008 provided high-resolution structures for only 28 of 1637 Type II REase sequences available in the Restriction Enzyme database (REBASE). Among these structures, all but two possess catalytic domains with the common PD-(D/E)XK nuclease fold. Two structures are unrelated to the others: R.BfiI exhibits the phospholipase D (PLD) fold, while R.PabI has a new fold termed 'half-pipe'. Thus far, bioinformatic studies supported by site-directed mutagenesis have extended the number of tentatively assigned REase folds to five (now including also GIY-YIG and HNH folds identified earlier in homing endonucleases) and provided structural predictions for dozens of REase sequences without experimentally solved structures. Here, we present a comprehensive study of all Type II REase sequences available in REBASE together with their homologs detectable in the nonredundant and environmental samples databases at the NCBI. We present the summary and critical evaluation of structural assignments and predictions reported earlier, new classification of all REase sequences into families, domain architecture analysis and new predictions of three-dimensional folds. Among 289 experimentally characterized (not putative) Type II REases, whose apparently full-length sequences are available in REBASE, we assign 199 (69%) to contain the PD-(D/E)XK domain. The HNH domain is the second most common, with 24 (8%) members. When putative REases are taken into account, the fraction of PD-(D/E)XK and HNH folds changes to 48% and 30%, respectively. Fifty-six characterized (and 521 predicted) REases remain unassigned to any of the five REase folds identified so far, and may exhibit new architectures. These enzymes are proposed as the most interesting targets for structure determination by high-resolution experimental methods. Our analysis provides the first comprehensive map of sequence-structure relationships among Type II REases and will help to focus the efforts of structural and functional genomics of this large and biotechnologically important class of enzymes.