Cellular localization of Type I restriction-modification enzymes is family dependent

Cellular localization of Type I restriction-modification enzymes EcoKI, EcoAI, and EcoR124I-the most frequently studied representatives of IA, IB, and IC families-was analyzed by immunoblotting of subcellular fractions isolated from Escherichia coli strains harboring the corresponding hsd genes. EcoR124I shows characteristics similar to those of EcoKI. The complex enzymes are associated with the cytoplasmic membrane via DNA interaction as documented by the release of the Hsd subunits from the membrane into the soluble fraction following benzonase treatment. HsdR subunits of the membrane-bound enzymes EcoKI and EcoR124I are accessible, though to a different extent, at the external surface of cytoplasmic membrane as shown by trypsinization of intact spheroplasts. EcoAI strongly differs from EcoKI and EcoR124I, since neither benzonase nor trypsin affects its association with the cytoplasmic membrane. Possible reasons for such a different organization are discussed in relation of the control of the restriction-modification activities in vivo.     

The HsdR subunit of R.EcoR124II: cloning and over-expression of the gene and unexpected properties of the subunit.

Type I restriction endonucleases are composed of three subunits, HsdR, HsdM and HsdS. The HsdR subunit is absolutely required for restriction activity; while an independent methylase is composed of HsdM and HsdS subunits. DNA cleavage is associated with a powerful ATPase activity during which DNA is translocated by the enzyme prior to cleavage. The presence of a Walker type I ATP-binding site within the HsdR subunit suggested that the subunit may be capable of independent enzymatic activity. Therefore, we have, for the first time, cloned and over-expressed the hsdRgene of the type IC restriction endonuclease EcoR124II. The purified HsdR subunit was found to be a soluble monomeric protein capable of DNA- and Mg2+-dependent ATP hydrolysis. The subunit was found to have a weak nuclease activity both in vivo and in vitro, and to bind plasmid DNA; although was not capable of binding a DNA oligoduplex. We were also able to reconstitute the fully active endonuclease from purified M. EcoR124I and HsdR. This is the first clear demonstration that the HsdR subunit of a type I restriction endonuclease is capable of independent enzyme activity, and suggests a mechanism for the evolution of the endonuclease from the independent methylase.     

Phosphorylation of Type IA restriction-modification complex enzyme EcoKI on the HsdR subunit

Phosphorylation of Type I restriction-modification (R-M) enzymes EcoKI, EcoAI, and EcoR124I - representatives of IA, IB, and IC families, respectively - was analysed in vivo by immunoblotting of endogenous phosphoproteins isolated from Escherichia coli strains harbouring the corresponding hsd genes, and in vitro by a phosphorylation assay using protein kinase present in subcellular fractions of E. coli. From all three R-M enzymes, the HsdR subunit of EcoKI system was the only subunit that was phosphorylated. Further, evidence is presented that HsdR is phosphorylated in vivo only when coproduced with HsdM and HsdS subunits - as part of assembled EcoKI restriction endonuclease, while the individually produced HsdR subunit is not phosphorylated. In vitro phosphorylation of the HsdR subunit of purified EcoKI endonuclease occurs on Thr, and is strictly dependent on the addition of a catalytic amount of cytoplasmic fraction isolated from E. coli. So far this is the first case of phosphorylation of a Type I R-M enzyme reported.     

Functional Coupling of Duplex Translocation to DNA Cleavage in a Type I Restriction Enzyme

Type I restriction-modification enzymes are multifunctional heteromeric complexes with DNA cleavage and ATP-dependent DNA translocation activities located on motor subunit HsdR. Functional coupling of DNA cleavage and translocation is a hallmark of the Type I restriction systems that is consistent with their proposed role in horizontal gene transfer. DNA cleavage occurs at nonspecific sites distant from the cognate recognition sequence, apparently triggered by stalled translocation. The X-ray crystal structure of the complete HsdR subunit from E. coli plasmid R124 suggested that the triggering mechanism involves interdomain contacts mediated by ATP. In the present work, in vivo and in vitro activity assays and crystal structures of three mutants of EcoR124I HsdR designed to probe this mechanism are reported. The results indicate that interdomain engagement via ATP is indeed responsible for signal transmission between the endonuclease and helicase domains of the motor subunit. A previously identified sequence motif that is shared by the RecB nucleases and some Type I endonucleases is implicated in signaling.     

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.     

An investigation of the structural requirements for ATP hydrolysis and DNA cleavage by the EcoKI Type I DNA restriction and modification enzyme

Type I DNA restriction/modification systems are oligomeric enzymes capable of switching between a methyltransferase function on hemimethylated host DNA and an endonuclease function on unmethylated foreign DNA. They have long been believed to not turnover as endonucleases with the enzyme becoming inactive after cleavage. Cleavage is preceded and followed by extensive ATP hydrolysis and DNA translocation. A role for dissociation of subunits to allow their reuse has been proposed for the EcoR124I enzyme. The EcoKI enzyme is a stable assembly in the absence of DNA, so recycling was thought impossible. Here, we demonstrate that EcoKI becomes unstable on long unmethylated DNA; reuse of the methyltransferase subunits is possible so that restriction proceeds until the restriction subunits have been depleted. We observed that RecBCD exonuclease halts restriction and does not assist recycling. We examined the DNA structure required to initiate ATP hydrolysis by EcoKI and find that a 21-bp duplex with single-stranded extensions of 12 bases on either side of the target sequence is sufficient to support hydrolysis. Lastly, we discuss whether turnover is an evolutionary requirement for restriction, show that the ATP hydrolysis is not deleterious to the host cell and discuss how foreign DNA occasionally becomes fully methylated by these systems.     

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.     

The interrelationship of helicase and nuclease domains during DNA translocation by the molecular motor EcoR124I

The type I restriction-modification enzyme EcoR124I comprises three subunits with the stoichiometry HsdR₂/HsdM₂/HsdS₁. The HsdR subunits are archetypical examples of the fusion between nuclease and helicase domains into a single polypeptide, a linkage that is found in a great many other DNA processing enzymes. To explore the interrelationship between these physically linked domains, we examined the DNA translocation properties of EcoR124I complexes in which the HsdR subunits had been mutated in the RecB-like nuclease motif II or III. We found that nuclease mutations can have multiple effects on DNA translocation despite being distinct from the helicase domain. In addition to reductions in DNA cleavage activity, we also observed decreased translocation and ATPase rates, different enzyme populations with different characteristic translocation rates, a tendency to stall during initiation and altered HsdR turnover dynamics. The significance of these observations to our understanding of domain interactions in molecular machines is discussed.     

DNA translocation blockage, a general mechanism of cleavage site selection by type I restriction enzymes

Type I restriction enzymes bind to a specific DNA sequence and subsequently translocate DNA past the complex to reach a non-specific cleavage site. We have examined several potential blocks to DNA translocation, such as positive supercoiling or a Holliday junction, for their ability to trigger DNA cleavage by type I restriction enzymes. Introduction of positive supercoiling into plasmid DNA did not have a significant effect on the rate of DNA cleavage by EcoAI endonuclease nor on the enzyme's ability to select cleavage sites randomly throughout the DNA molecule. Thus, positive supercoiling does not prevent DNA translocation. EcoR124II endonuclease cleaved DNA at Holliday junctions present on both linear and negatively supercoiled substrates. The latter substrate was cleaved by a single enzyme molecule at two sites, one on either side of the junction, consistent with a bi-directional translocation model. Linear DNA molecules with two recognition sites for endonucleases from different type I families were cut between the sites when both enzymes were added simultaneously but not when a single enzyme was added. We propose that type I restriction enzymes can track along a DNA substrate irrespective of its topology and cleave DNA at any barrier that is able to halt the translocation process.     

The type I restriction endonuclease EcoR124I, couples ATP hydrolysis to bidirectional DNA translocation

Type I restriction endonuclease holoenzymes contain methylase (M), restriction (R) and specificity (S) subunits, present in an M2:R2:S1 stoichiometry. These enzymes bind to specific DNA sequences and translocate dsDNA in an ATP-dependent manner toward the holoenzyme anchored at the recognition sequence. Once translocation is impeded, DNA restriction, which functions to protect the host cell from invading DNA, takes place. Translocation and DNA cleavage are afforded by the two diametrically opposed R-subunits. To gain insight into the mechanism of translocation, a detailed characterization of the ATPase activity of EcoR124I was done. Results show that following recognition sequence binding, ATP hydrolysis-coupled, bidirectional DNA translocation by EcoR124I ensues, with the R-subunits transiently disengaging, on average, every 515 bp. Macroscopic processivity of 2031(+/-184)bp is maintained, as the R-subunits remain in close proximity to the DNA through association with the methyltransferase. Transient uncoupling of ATP hydrolysis from translocation results in 3.1(+/-0.4) ATP molecules being hydrolyzed per base-pair translocated per R-subunit. This is the first clear demonstration of the coupling of ATP hydrolysis to dsDNA translocation, albeit inefficient. Once translocation is impeded on supercoiled DNA, the DNA is cleaved. DNA cleavage inactivates the EcoR124I holoenzyme partially and reversibly, which explains the stoichiometric behaviour of type I restriction enzymes. Inactivated holoenzyme remains bound to the DNA at the recognition sequence and immediately releases the nascent ends. The release of nascent ends was demonstrated using a novel, fluorescence-based, real-time assay that takes advantage of the ability of the Escherichia coli RecBCD enzyme to unwind restricted dsDNA. The resulting unwinding of EcoR124I-restricted DNA by RecBCD reveals coordination between the restriction-modification and recombination systems that functions to destroy invading DNA efficiently. In addition, we demonstrate the displacement of EcoR124I following DNA cleavage by the translocating RecBCD enzyme, resulting in the restoration of catalytic function to EcoR124I.     

Real-time observation of DNA translocation by the type I restriction modification enzyme EcoR124I

Type I restriction enzymes bind sequence-specifically to unmodified DNA and subsequently pull the adjacent DNA toward themselves. Cleavage then occurs remotely from the recognition site. The mechanism by which these members of the superfamily 2 (SF2) of helicases translocate DNA is largely unknown. We report the first single-molecule study of DNA translocation by the type I restriction enzyme EcoR124I. Mechanochemical parameters such as the translocation rate and processivity, and their dependence on force and ATP concentration, are presented. We show that the two motor subunits of EcoR124I work independently. By using torsionally constrained DNA molecules, we found that the enzyme tracks along the helical pitch of the DNA molecule. This assay may be directly applicable to investigating the tracking of other DNA-translocating motors along their DNA templates.     

Measuring motion on DNA by the type I restriction endonuclease EcoR124I using triplex displacement

The type I restriction enzyme EcoR124I cleaves DNA following extensive linear translocation dependent upon ATP hydrolysis. Using protein-directed displacement of a DNA triplex, we have determined the kinetics of one-dimensional motion without the necessity of measuring DNA or ATP hydrolysis. The triplex was pre-formed specifically on linear DNA, 4370 bp from an EcoR124I site, and then incubated with endonuclease. Upon ATP addition, a distinct lag phase was observed before the triplex-forming oligonucleotide was displaced with exponential kinetics. As the distance between type I and triplex sites was shortened, the lag time decreased whilst the displacement reaction remained exponential. This is indicative of processive DNA translocation followed by collision with the triplex and oligonucleotide displacement. A linear relationship between lag duration and inter-site distance gives a translocation velocity of 400+/-32 bp/s at 20 degrees C. Furthermore, the data can only be explained by bi-directional translocation. An endonuclease with only one of the two HsdR subunits responsible for motion could still catalyse translocation. The reaction is less processive, but can 'reset' in either direction whenever the DNA is released.     

Repercussions of DNA tracking by the type IC restriction endonuclease EcoR124I on linear, circular and catenated substrates.

Type I restriction endonucleases such as EcoR124I cleave DNA at undefined loci, distant from their recognition sequences, by a mechanism that involves the enzyme tracking along the DNA between recognition and cleavage sites. This mechanism was examined on plasmids that carried recognition sites for EcoR124I and recombination sites for resolvase, the latter to create DNA catenanes. Supercoiled substrates with either one or two restriction sites were linearized by EcoR124I at similar rates, although the two-site molecule underwent further cleavage more readily than the one-site DNA. The catenane from the plasmid with one EcoR124I site, carrying the site on the smaller of the two rings, was cleaved by EcoR124I exclusively in the small ring, and this underwent multiple cleavage akin to the two-site plasmid. Linear substrates derived from the plasmids were cleaved by EcoR124I at very slow rates. The communication between recognition and cleavage sites therefore cannot stem from random looping. Instead, it must follow the DNA contour between the sites. On a circular DNA, the translocation of non-specific DNA past the specifically bound protein should increase negative supercoiling in one domain and decrease it in the other. The ensuing topological barrier may be the trigger for DNA cleavage.     

A novel mutant of the type I restriction-modification enzyme EcoR124I is altered at a key stage of the subunit assembly pathway

The HsdS subunit of a type I restriction-modification (R-M) system plays an essential role in the activity of both the modification methylase and the restriction endonuclease. This subunit is responsible for DNA binding, but also contains conserved amino acid sequences responsible for protein-protein interactions. The most important protein-protein interactions are those between the HsdS subunit and the HsdM (methylation) subunit that result in assembly of an independent methylase (MTase) of stoichiometry M(2)S(1). Here, we analysed the impact on the restriction and modification activities of the change Trp(212)-->Arg in the distal border of the central conserved region of the EcoR124I HsdS subunit. We demonstrate that this point mutation significantly influences the ability of the mutant HsdS subunit to assemble with the HsdM subunit to produce a functional MTase. As a consequence of this, the mutant MTase has drastically reduced DNA binding, which is restored only when the HsdR (restriction) subunit binds with the MTase. Therefore, HsdR acts as a chaperon allowing not only binding of the enzyme to DNA, but also restoring the methylation activity and, at sufficiently high concentrations in vitro of HsdR, restoring restriction activity.     

A residue of motif III positions the helicase domains of motor subunit HsdR in restriction-modification enzyme EcoR124I

Type I restriction-modification enzymes differ significantly from the type II enzymes commonly used as molecular biology reagents. On hemi-methylated DNAs type I enzymes like the EcoR124I restriction-modification complex act as conventional adenine methylases at their specific target sequences, but unmethylated targets induce them to translocate thousands of base pairs through the stationary enzyme before cleaving distant sites nonspecifically. EcoR124I is a superfamily 2 DEAD-box helicase like eukaryotic double-strand DNA translocase Rad54, with two RecA-like helicase domains and seven characteristic sequence motifs that are implicated in translocation. In Rad54 a so-called extended region adjacent to motif III is involved in ATPase activity. Although the EcoR124I extended region bears sequence and structural similarities with Rad54, it does not influence ATPase or restriction activity as shown in this work, but mutagenesis of the conserved glycine residue of its motif III does alter ATPase and DNA cleavage activity. Through the lens of molecular dynamics, a full model of HsdR of EcoR124I based on available crystal structures allowed interpretation of functional effects of mutants in motif III and its extended region. The results indicate that the conserved glycine residue of motif III has a role in positioning the two helicase domains.     

Localization of the type I restriction-modification enzyme EcoKI in the bacterial cell

To localise the type I restriction-modification (R-M) enzyme EcoKI within the bacterial cell, the Hsd subunits present in subcellular fractions were analysed using immunoblotting techniques. The endonuclease (ENase) as well as the methylase (MTase) were found to be associated with the cytoplasmic membrane. HsdR and HsdM subunits produced individually were soluble, cytoplasmic polypeptides and only became membrane-associated when coproduced with the insoluble HsdS subunit. The release of enzyme from the membrane fraction following benzonase treatment indicated a role for DNA in this interaction. Trypsinization of spheroplasts revealed that the HsdR subunit in the assembled ENase was accessible to protease, while HsdM and HsdS, in both ENase and MTase complexes, were fully protected against digestion. We postulate that the R-M enzyme EcoKI is associated with the cytoplasmic membrane in a manner that allows access of HsdR to the periplasmic space, while the MTase components are localised on the inner side of the plasma membrane.     

Initiation of translocation by Type I restriction-modification enzymes is associated with a short DNA extrusion

Recognition of ‘foreign’ DNA by Type I restriction–modification (R-M) enzymes elicits an ATP-dependent switch from methylase to endonuclease activity, which involves DNA translocation by the restriction subunit HsdR. Type I R-M enzymes are composed of three (Hsd) subunits with a stoichiometry of HsdR₂:HsdM₂:HsdS₁ (R₂-complex). However, the EcoR124I R-M enzyme can also exist as a cleavage deficient, sub-assembly of HsdR₁:HsdM₂:HsdS₁ (R₁-complex). ATPγS was used to trap initial translocation complexes, which were visualized by Atomic Force Microscopy (AFM). In the R₁-complex, a small bulge, associated with a shortening in the contour-length of the DNA of 8 nm, was observed. This bulge was found to be sensitive to single-strand DNA nucleases, indicative of non-duplexed DNA. R₂-complexes appeared larger in the AFM images and the DNA contour length showed a shortening of ~11 nm, suggesting that two bulges were formed. Disclosure of the structure of the first stage after the recognition-translocation switch of Type I restriction enzymes forms an important first step in resolving a detailed mechanistic picture of DNA translocation by SF-II DNA translocation motors.     

Dynamics of initiation, termination and reinitiation of DNA translocation by the motor protein EcoR124I

Type I restriction enzymes use two motors to translocate DNA before carrying out DNA cleavage. The motor function is accomplished by amino-acid motifs typical for superfamily 2 helicases, although DNA unwinding is not observed. Using a combination of extensive single-molecule magnetic tweezers and stopped-flow bulk measurements, we fully characterized the (re)initiation of DNA translocation by EcoR124I. We found that the methyltransferase core unit of the enzyme loads the motor subunits onto adjacent DNA by allowing them to bind and initiate translocation. Termination of translocation occurs owing to dissociation of the motors from the core unit. Reinitiation of translocation requires binding of new motors from solution. The identification and quantification of further initiation steps--ATP binding and extrusion of an initial DNA loop--allowed us to deduce a complete kinetic reinitiation scheme. The dissociation/reassociation of motors during translocation allows dynamic control of the restriction process by the availability of motors. Direct evidence that this control mechanism is relevant in vivo is provided.     

Structural and functional analysis of the symmetrical Type I restriction endonuclease R.EcoR124I(NT)

Type I restriction-modification (RM) systems are comprised of two multi-subunit enzymes, the methyltransferase (～160 kDa), responsible for methylation of DNA, and the restriction endonuclease (～400 kDa), responsible for DNA cleavage. Both enzymes share a number of subunits. An engineered RM system, EcoR124I(NT), based on the N-terminal domain of the specificity subunit of EcoR124I was constructed that recognises the symmetrical sequence GAAN(7)TTC and is active as a methyltransferase. Here, we investigate the restriction endonuclease activity of R. EcoR124I(NT)in vitro and the subunit assembly of the multi-subunit enzyme. Finally, using small-angle neutron scattering and selective deuteration, we present a low-resolution structural model of the endonuclease and locate the motor subunits within the multi-subunit enzyme. We show that the covalent linkage between the two target recognition domains of the specificity subunit is not required for subunit assembly or enzyme activity, and discuss the implications for the evolution of Type I enzymes.     

When a helicase is not a helicase: dsDNA tracking by the motor protein EcoR124I

Using a combination of single molecule and bulk solution measurements, we have examined the DNA translocation activity of a helicase, the Type I restriction modification enzyme EcoR124I. We find that EcoR124I can translocate past covalent interstrand crosslinks, inconsistent with an obligatory unwinding mechanism. Instead, translocation of the intact dsDNA occurs principally via contacts to the sugar-phosphate backbone and bases of the 3'-5' strand; contacts to the 5'-3' strand are not essential for motion but do play a key role in stabilising the motor on the DNA. A model for dsDNA translocation is presented that could be applicable to a wide range of other enzyme complexes that are also labelled as helicases but which do not have actual unwinding activity.