Translocation-independent dimerization of the EcoKI endonuclease visualized by atomic force microscopy

Bacterial type I restriction/modification systems are capable of performing multiple actions in response to the methylation pattern on their DNA recognition sequences. The enzymes making up these systems serve to protect the bacterial cells against viral infection by binding to their recognition sequences on the invading DNA and degrading it after extensive ATP-driven translocation. DNA cleavage has been thought to occur as the result of a collision between two translocating enzyme complexes. Using atomic force microscopy (AFM), we show here that EcoKI dimerizes rapidly when bound to a plasmid containing two recognition sites for the enzyme. Dimerization proceeds in the absence of ATP and is also seen with an EcoKI mutant (K477R) that is unable to translocate DNA. Only monomers are seen when the enzyme complex binds to a plasmid containing a single recognition site. Based on our results, we propose that the binding of EcoKI to specific DNA target sequences is accompanied by a conformational change that leads rapidly to dimerization. This event is followed by ATP-dependent translocation and cleavage of the DNA.     

The structure of M.EcoKI Type I DNA methyltransferase with a DNA mimic antirestriction protein

Type-I DNA restriction–modification (R/M) systems are important agents in limiting the transmission of mobile genetic elements responsible for spreading bacterial resistance to antibiotics. EcoKI, a Type I R/M enzyme from Escherichia coli, acts by methylation- and sequence-specific recognition, leading to either methylation of DNA or translocation and cutting at a random site, often hundreds of base pairs away. Consisting of one specificity subunit, two modification subunits, and two DNA translocase/endonuclease subunits, EcoKI is inhibited by the T7 phage antirestriction protein ocr, a DNA mimic. We present a 3D density map generated by negative-stain electron microscopy and single particle analysis of the central core of the restriction complex, the M.EcoKI M₂S₁ methyltransferase, bound to ocr. We also present complete atomic models of M.EcoKI in complex with ocr and its cognate DNA giving a clear picture of the overall clamp-like operation of the enzyme. The model is consistent with a large body of experimental data on EcoKI published over 40 years.     

Plasmid-encoded antirestriction protein ArdA can discriminate between type I methyltransferase and complete restriction-modification system

Many promiscuous plasmids encode the antirestriction proteins ArdA (alleviation of restriction of DNA) that specifically affect the restriction activity of heterooligomeric type I restriction-modification (R-M) systems in Escherichia coli cells. In addition, a lot of the putative ardA genes encoded by plasmids and bacterial chromosomes are found as a result of sequencing of complete genomic sequences, suggesting that ArdA proteins and type I R-M systems that seem to be widespread among bacteria may be involved in the regulation of gene transfer among bacterial genomes. Here, the mechanism of antirestriction action of ArdA encoded by IncI plasmid ColIb-P9 has been investigated in comparison with that of well-studied T7 phage-encoded antirestriction protein Ocr using the mutational analysis, retardation assay and His-tag affinity chromatography. Like Ocr, ArdA protein was shown to be able to efficiently interact with EcoKI R-M complex and affect its in vivo and in vitro restriction activity by preventing its interaction with specific DNA. However, unlike Ocr, ArdA protein has a low binding affinity to EcoKI Mtase and the additional C-terminal tail region (VF-motif) is needed for ArdA to efficiently interact with the type I R-M enzymes. It seems likely that this ArdA feature is a basis for its ability to discriminate between activities of EcoKI Mtase (modification) and complete R-M system (restriction) which may interact with unmodified DNA in the cells independently. These findings suggest that ArdA may provide a very effective and delicate control for the restriction and modification activities of type I systems and its ability to discriminate against DNA restriction in favour of the specific modification of DNA may give some advantage for efficient transmission of the ardA-encoding promiscuous plasmids among different bacterial populations.     

Lytic Replication of Coliphage Lambda in Salmonella typhosa Hybrids

Hybrids between Escherichia coli K-12 and Salmonella typhosa which conserved a continuous K-12 chromosomal diploid segment extending from pro through ara to the strA locus were sensitive to plaque formation by wild-type λ. These partially diploid S. typhosa hybrids could be lysogenized with λ and subsequently induced to produce infectious phage particles. When the K-12 genes were segregated from a lysogenic S. typhosa hybrid, phage-productive ability was no longer detectable due to loss of a genetic region necessary for vegetative replication of λ. However, λ prophage was shown to persist in a quiescent state in the S. typhosa hybrid segregant with phage-productive ability being reactivated after replacement of the essential K-12 λ replication region. Low-frequency transduction and high-frequency transduction lysates containing the gal⁺ genes of S. typhosa were prepared by induction of λ-lysogenic S. typhosa hybrids indicating that the attλ site is chromosomally located in S. typhosa in close proximity to the gal locus as in E. coli K-12. After propagation in S. typhosa hybrids, λ was subject to restriction by E. coli K-12 recipients, thus establishing that S. typhosa does not perform the K-12 modification of λ deoxyribonucleic acid. Hybrids of S. typhosa, however, did not restrict λ grown previously on E. coli K-12. The K-12 genetic region required for λ phage production in S. typhosa was located within min 66 to min 72 on the genetic map of the E. coli chromosome. Transfer of an F-merogenote encompassing the 66 to 72 min E. coli chromosomal region to λ-insensitive S. typhosa hybrids enabled them to replicate wild-type λ. The λ-insensitive S. typhosa hybrid, WR4255, which blocks λ replication, can be mutagenized to yield mutant strains sensitive to λvir and λimm434. These WR4255 mutants remained insensitive to plaque formation by wild-type λ.     

StpA protein from Escherichia coli condenses supercoiled DNA in preference to linear DNA and protects it from digestion by DNase I and EcoKI

The nucleoid-associated protein, StpA, of Escherichia coli binds non-specifically to double-stranded DNA (dsDNA) and apparently forms bridges between adjacent segments of the DNA. Such a coating of protein on the DNA would be expected to hinder the action of nucleases. We demonstrate that StpA binding hinders dsDNA cleavage by both the non-specific endonuclease, DNase I, and by the site-specific type I restriction endonuclease, EcoKI. It requires approximately one StpA molecule per 250–300 bp of supercoiled DNA and approximately one StpA molecule per 60–100 bp on linear DNA for strong inhibition of the nucleases. These results support the role of StpA as a nucleoid-structuring protein which binds DNA segments together. The inhibition of EcoKI, which cleaves DNA at a site remote from its initial target sequence after extensive DNA translocation driven by ATP hydrolysis, suggests that these enzymes would be unable to function on chromosomal DNA even during times of DNA damage when potentially lethal, unmodified target sites occur on the chromosome. This supports a role for nucleoid-associated proteins in restriction alleviation during times of cell stress.     

Characterisation of the catalytically active form of RecG helicase

Replication of DNA is fraught with difficulty and chromosomes contain many lesions which may block movement of the replicative machinery. However, several mechanisms to overcome such problems are beginning to emerge from studies with Escherichia coli. An important enzyme in one or more of these mechanisms is the RecG helicase, which may target stalled replication forks to generate a four-stranded (Holliday) junction, thus facilitating repair and/or bypass of the original lesion. To begin to understand how RecG might catalyse regression of fork structures, we have analysed what the catalytically active form of the enzyme may be. We have found that RecG exists as a monomer in solution as measured by gel filtration but when bound to junction DNA the enzyme forms two distinct protein–DNA complexes that contain one and two protein molecules. However, mutant inhibition studies failed to provide any evidence that RecG acts as a multimer in vitro. Additionally, there was no evidence for cooperativity in the junction DNA-stimulated hydrolysis of ATP. These data suggest that RecG functions as a monomer to unwind junction DNA, which supports an ‘inchworm’ rather than an ‘active rolling’ mechanism of DNA unwinding. The observed in vivo inhibition of wild-type RecG by mutant forms of the enzyme was attributed to occlusion of the DNA target and correlates with the very low abundance of replication forks within an E.coli cell, even during rapid growth.     

Removal of a frameshift between the hsdM and hsdS genes of the EcoKI Type IA DNA restriction and modification system produces a new type of system and links the different families of Type I systems

The EcoKI DNA methyltransferase is a trimeric protein comprised of two modification subunits (M) and one sequence specificity subunit (S). This enzyme forms the core of the EcoKI restriction/modification (RM) enzyme. The 3′ end of the gene encoding the M subunit overlaps by 1 nt the start of the gene for the S subunit. Translation from the two different open reading frames is translationally coupled. Mutagenesis to remove the frameshift and fuse the two subunits together produces a functional RM enzyme in vivo with the same properties as the natural EcoKI system. The fusion protein can be purified and forms an active restriction enzyme upon addition of restriction subunits and of additional M subunit. The Type I RM systems are grouped into families, IA to IE, defined by complementation, hybridization and sequence similarity. The fusion protein forms an evolutionary intermediate form lying between the Type IA family of RM enzymes and the Type IB family of RM enzymes which have the frameshift located at a different part of the gene sequence.     

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.     

A Mimicking-of-DNA-Methylation-Patterns Pipeline for Overcoming the Restriction Barrier of Bacteria

Genetic transformation of bacteria harboring multiple Restriction-Modification (R-M) systems is often difficult using conventional methods. Here, we describe a mimicking-of-DNA-methylation-patterns (MoDMP) pipeline to address this problem in three difficult-to-transform bacterial strains. Twenty-four putative DNA methyltransferases (MTases) from these difficult-to-transform strains were cloned and expressed in an Escherichia coli strain lacking all of the known R-M systems and orphan MTases. Thirteen of these MTases exhibited DNA modification activity in Southwestern dot blot or Liquid Chromatography–Mass Spectrometry (LC–MS) assays. The active MTase genes were assembled into three operons using the Saccharomyces cerevisiae DNA assembler and were co-expressed in the E. coli strain lacking known R-M systems and orphan MTases. Thereafter, results from the dot blot and restriction enzyme digestion assays indicated that the DNA methylation patterns of the difficult-to-transform strains are mimicked in these E. coli hosts. The transformation of the Gram-positive Bacillus amyloliquefaciens TA208 and B. cereus ATCC 10987 strains with the shuttle plasmids prepared from MoDMP hosts showed increased efficiencies (up to four orders of magnitude) compared to those using the plasmids prepared from the E. coli strain lacking known R-M systems and orphan MTases or its parental strain. Additionally, the gene coding for uracil phosphoribosyltransferase (upp) was directly inactivated using non-replicative plasmids prepared from the MoDMP host in B. amyloliquefaciens TA208. Moreover, the Gram-negative chemoautotrophic Nitrobacter hamburgensis strain X14 was transformed and expressed Green Fluorescent Protein (GFP). Finally, the sequence specificities of active MTases were identified by restriction enzyme digestion, making the MoDMP system potentially useful for other strains. The effectiveness of the MoDMP pipeline in different bacterial groups suggests a universal potential. This pipeline could facilitate the functional genomics of the strains that are difficult to transform.     

Type I restriction systems: sophisticated molecular machines (a legacy of Bertani and Weigle).

Restriction enzymes are well known as reagents widely used by molecular biologists for genetic manipulation and analysis, but these reagents represent only one class (type II) of a wider range of enzymes that recognize specific nucleotide sequences in DNA molecules and detect the provenance of the DNA on the basis of specific modifications to their target sequence. Type I restriction and modification (R-M) systems are complex; a single multifunctional enzyme can respond to the modification state of its target sequence with the alternative activities of modification or restriction. In the absence of DNA modification, a type I R-M enzyme behaves like a molecular motor, translocating vast stretches of DNA towards itself before eventually breaking the DNA molecule. These sophisticated enzymes are the focus of this review, which will emphasize those aspects that give insights into more general problems of molecular and microbial biology. Current molecular experiments explore target recognition, intramolecular communication, and enzyme activities, including DNA translocation. Type I R-M systems are notable for their ability to evolve new specificities, even in laboratory cultures. This observation raises the important question of how bacteria protect their chromosomes from destruction by newly acquired restriction specifities. Recent experiments demonstrate proteolytic mechanisms by which cells avoid DNA breakage by a type I R-M system whenever their chromosomal DNA acquires unmodified target sequences. Finally, the review will reflect the present impact of genomic sequences on a field that has previously derived information almost exclusively from the analysis of bacteria commonly studied in the laboratory.     

The EcoKI Type I Restriction-Modification System in Escherichia coli Affects but Is Not an Absolute Barrier for Conjugation

The rapid evolution of bacteria is crucial to their survival and is caused by exchange, transfer, and uptake of DNA, among other things. Conjugation is one of the main mechanisms by which bacteria share their DNA, and it is thought to be controlled by varied bacterial immune systems. Contradictory results about restriction-modification systems based on phenotypic studies have been presented as reasons for a barrier to conjugation with and other means of uptake of exogenous DNA. In this study, we show that inactivation of the R.EcoKI restriction enzyme in strain Escherichia coli K-12 strain MG1655 increases the conjugational transfer of plasmid pOLA52, which carriers two EcoKI recognition sites. Interestingly, the results were not absolute, and uptake of unmethylated pOLA52 was still observed in the wild-type strain (with an intact hsdR gene) but at a reduction of 85% compared to the uptake of the mutant recipient with a disrupted hsdR gene. This leads to the conclusion that EcoKI restriction-modification affects the uptake of DNA by conjugation but is not a major barrier to plasmid transfer.     

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.     

MmeI: a minimal Type II restriction-modification system that only modifies one DNA strand for host protection

MmeI is an unusual Type II restriction enzyme that is useful for generating long sequence tags. We have cloned the MmeI restriction-modification (R-M) system and found it to consist of a single protein having both endonuclease and DNA methyltransferase activities. The protein comprises an amino-terminal endonuclease domain, a central DNA methyltransferase domain and C-terminal DNA recognition domain. The endonuclease cuts the two DNA strands at one site simultaneously, with enzyme bound at two sites interacting to accomplish scission. Cleavage occurs more rapidly than methyl transfer on unmodified DNA. MmeI modifies only the adenine in the top strand, 5′-TCCRAC-3′. MmeI endonuclease activity is blocked by this top strand adenine methylation and is unaffected by methylation of the adenine in the complementary strand, 5′-GTYGGA-3′. There is no additional DNA modification associated with the MmeI R-M system, as is required for previously characterized Type IIG R-M systems. The MmeI R-M system thus uses modification on only one of the two DNA strands for host protection. The MmeI architecture represents a minimal approach to assembling a restriction-modification system wherein a single DNA recognition domain targets both the endonuclease and DNA methyltransferase activities.     

Host Specificity of Salmonella typhimurium Deoxyribonucleic Acid Restriction and Modification

The restriction and modification genes of Salmonella typhimurium which lie near the thr locus were transferred to a restrictionless mutant of Escherichia coli. These genes were found to be allelic to the E. coli K, B, and A restriction and modification genes. E. coli recombinants with the restriction and modification host specificity of S. typhimurium restricted phage λ that had been modified by each of the seven known host specificities of E. coli at efficiency of plating levels of about 10⁻². Phage λ modified with the S. typhimurium host specificity was restricted by six of the seven E. coli host specificities but not by the RII (fi⁻ R-factor controlled) host specificity. It is proposed that the restriction and modification enzymes of this S. typhimurium host specificity have two substrates, one of which is a substrate for the RII host specificity enzymes.     

Haemophilus influenzae phasevarions have evolved from type III DNA restriction systems into epigenetic regulators of gene expression

Phase variably expressed (randomly switching) methyltransferases associated with type III restriction-modification (R-M) systems have been identified in a variety of pathogenic bacteria. We have previously shown that a phase variable methyltransferase (Mod) associated with a type III R-M system in Haemophilus influenzae strain Rd coordinates the random switching of expression of multiple genes, and constitutes a phase variable regulon—‘phasevarion’. We have now identified the recognition site for the Mod methyltransferase in H. influenzae strain Rd as 5′-CGAAT-3′. This is the same recognition site as the previously described HinfIII system. A survey of 59 H. influenzae strains indicated significant sequence heterogeneity in the central, variable region of the mod gene associated with target site recognition. Intra- and inter-strain transformation experiments using Mod methylated or non-methylated plasmids, and a methylation site assay demonstrated that the sequence heterogeneity seen in the region encoding target site specificity does correlate to distinct target sites. Mutations were identified within the res gene in several strains surveyed indicating that Res is not functional. These data suggest that evolution of this type III R-M system into an epigenetic mechanism for controlling gene expression has, in some strains, resulted in loss of the DNA restriction function.     

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.     

High-throughput,cost-effective verification of structural DNA assembly

DNA ‘assembly’ from ‘building blocks’ remains a cornerstone in synthetic biology, whether it be for gene synthesis (∼1 kb), pathway engineering (∼10 kb) or synthetic genomes (>100 kb). Despite numerous advances in the techniques used for DNA assembly, verification of the assembly is still a necessity, which becomes cost-prohibitive and a logistical challenge with increasing scale. Here we describe for the first time a comprehensive, high-throughput solution for structural DNA assembly verification by restriction digest using exhaustive in silico enzyme screening, rolling circle amplification of plasmid DNA, capillary electrophoresis and automated digest pattern recognition. This low-cost and robust methodology has been successfully used to screen over 31 000 clones of DNA constructs at <$1 per sample.     

Temperature-Sensitive Modification and Restriction Phenotypes of an Escherichia coli dnaD Mutant

A mutant of Escherichia coli temperature-sensitive for deoxyribonucleic acid synthesis, dnaD, was found to have temperature-sensitive modification and restriction phenotypes. In contrast to the original observation by Carl (1970), the mutant could support the growth of λ phage at 41 C. However, the λ phages thus produced were able to form plaques with normal plating efficiency only on E. coli C, a restriction-less strain, but not on E. coli K. Since the λ phages produced in the mutant at 30 C could form plaques equally well on both E. coli strains, it was concluded that the dnaD mutant has a temperature-sensitive modification phenotype. Furthermore, since the dnaD mutant allowed some growth of unmodified λ·C phages at 41 C but less at 30 C, the mutant is also temperature sensitive in restriction. The relationship, if any, between temperature-sensitive deoxyribonucleic acid synthesis and temperature-sensitive modification-restriction in the dnaD mutant is not known. Similar experiments were done with three dnaC mutants and one dnaA mutant. Two dnaC mutants were found to have altered restriction phenotypes at 41 C, but none of the mutants were defective in modification.     

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.