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.     

Basis for changes in DNA recognition by the EcoR124 and EcoR124/3 type I DNA restriction and modification enzymes

EcoR124 and EcoR124/3 are type I DNA restriction and modification systems. The EcoR124/3 system arose from the EcoR124 system some 15 years ago and at the electron microscopic DNA heteroduplex level the genes for both systems are still apparently identical. We have shown that the DNA sequences recognized by the two systems are GAA(N6)RTCG for EcoR124 and GAA(N7)RTCG for EcoR124/3. The sequences thus differ only in the length of the non-specific spacer. This difference nevertheless places the two specific domains of the EcoR124/3 recognition sequence 0.34 nm further apart and rotates them 36 degrees with respect to those of EcoR124, which implies major structural differences in the proteins recognizing these sequences. We have now determined the nucleotide sequences of the hsdS and hsdM genes of both systems and of the hsdR gene of EcoR124/3. The hsdS gene products provide DNA sequence specificity in both restriction and modification, the hsdM gene products are necessary for modification and all three hsd gene products are required for restriction. The only difference that we have detected between the two systems is that a 12 base-pair sequence towards the middle of the hsdS gene is repeated twice in the EcoR124 gene and three times in the EcoR124/3 gene. We have deleted one of the repeats in the EcoR124/3 gene and shown that this changes the specificity to that of EcoR124. Thus, the extra four amino acids in the middle of the EcoR124/3 hsdS gene product, which in an alpha-helical configuration would extend 0.6 nm, are sufficient to explain the differences in sequence recognition. We suggest that the EcoR124/3 system was generated by an unequal crossing over and argue that this kind of specificity change should not be rare in Nature.     

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.     

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.     

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.     

A motif conserved among the type I restriction-modification enzymes and antirestriction proteins: a possible basis for mechanism of action of plasmid-encoded antirestriction functions.

Antirestriction proteins Ard encoded by some self-transmissible plasmids specifically inhibit restriction by members of all three families of type I restriction-modification (R-M) systems in E.coli. Recently, we have identified the amino acid region, 'antirestriction' domain, that is conserved within different plasmid and phage T7-encoded antirestriction proteins and may be involved in interaction with the type I R-M systems. In this paper we demonstrate that this amino acid sequence shares considerable similarity with a well-known conserved sequence (the Argos repeat) found in the DNA sequence specificity (S) polypeptides of type I systems. We suggest that the presence of these similar motifs in restriction and antirestriction proteins may give a structural basis for their interaction and that the antirestriction action of Ard proteins may be a result of the competition between the 'antirestriction' domains of Ard proteins and the similar conserved domains of the S subunits that are believed to play a role in the subunit assembly of type I R-M systems.     

The EcoDXX1 restriction and modification system: cloning the genes and homology to type I restriction and modification systems

The Escherichia coli plasmid pDXX1 codes for a type I restriction and modification system, EcoDXX1. A 15.5-kb BamHI fragment from pDXX1 has been cloned and contains the hsdR, hsdM, and hsdS genes that encode the EcoDXX1 system. The EcoDXX1 hsd genes can complement the gene products of the EcoR124 and EcoR124/3 hsd systems, but not those of EcoK and EcoB. Hybridization experiments using EcoDXX1 hsd genes as a probe demonstrate homology between EcoDXX1 and EcoR124 and EcoR124/3 restriction-modification systems, but weak or no homology between EcoDXX1 and EcoK or EcoB systems.     

Recombination of constant and variable modules alters DNA sequence recognition by type IC restriction-modification enzymes.

EcoR124 and EcoDXXI are allelic type I restriction-modification (R-M) systems whose specificity genes consist of common structural elements: two variable regions are separated by a constant, homologous region containing a number of repetitive sequence elements. In vitro recombination of variable and constant elements has led to fully active, hybrid R-M systems exhibiting new and predictable target site specificities. Methylation of synthetic DNA sequences with purified, hybrid modification methylases was used to confirm the proposed recognition sequences. The results clearly demonstrate the correlation between protein domains and target site specificity. Our data suggest that a bacterial population may switch the recognition sequences of its type I R-M system by single recombination events and thus is able to maintain a prokaryotic analogue of the immune system of variable specificity.     

Characterization of AloI, a restriction-modification system of a new type.

We report the properties of the new AloI restriction and modification enzyme from Acinetobacter lwoffi Ks 4-8 that recognizes the DNA target 5' GGA(N)6GTTC3' (complementary strand 5' GAAC(N)6TCC3'), and the nucleotide sequence of the gene encoding this enzyme. AloI is a bifunctional large polypeptide (deduced M(r) 143 kDa) revealing both DNA endonuclease and methyltransferase activities. Depending on reaction cofactors, AloI cleaves double-stranded DNA on both strands, seven bases on the 5' side, and 12-13 bases on the 3' side of its recognition sequence, and modifies adenine residues in both DNA strands in the target sequence yielding N6-methyladenine. For cleavage activity AloI maintains an absolute requirement for Mg(2+) and does not depend on or is stimulated by either ATP or S-adenosyl-L-methionine. Modification function requires the presence of S-adenosyl-L-methionine and is stimulated by metal ions (Ca(2+)). The C-terminal and central parts of the protein were found to be homologous to certain specificity (HsdS) and modification (HsdM) subunits of type I R-M systems, respectively. The N-terminal part of the protein possesses the putative endonucleolytic motif DXnEXK of restriction endonucleases. The deduced amino acid sequence of AloI shares significant homology with polypeptides encoding HaeIV and CjeI restriction-modification proteins at the N-terminal and central, but not at the C-terminal domains. The organization of AloI implies that its evolution involved fusion of an endonuclease and the two subunits, HsdM and HsdS, of type I restriction enzymes. According to the structure and function properties AloI may be regarded as one more representative of a newly emerging group of HaeIV-like restriction endonucleases. Discovery of these enzymes opens new opportunities for constructing restriction endonucleases with a new specificity.     

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.     

A deletion mutant of the type IC restriction endonuclease EcoR1241 expressing a novel DNA specificity.

We have developed a complementation assay which allows us to distinguish between mutations affecting subunit assembly and mutations affecting DNA binding in the DNA recognition subunit (HsdS) of the multimeric restriction endonuclease EcoR1241. A number of random point mutations were constructed to test the validity of this assay. Two of the mutants produced were found to be truncated polypeptides that were still capable of complementation with the EcoR1241 Hsd subunits to give an active restriction enzyme of novel DNA specificity. The N-terminal variable domain (responsible for recognition of GAA from the EcoR1241 recognition sequence GAAnnnnnnRTCG) and the spacer region (central conserved region) is intact in both of these mutants. One of these mutant genes (hsdS(delta 50) has been cloned as an active Mtase. Purification of the Mtase proved to be difficult because the complex is weak. However, Mtase activity was obtained from a soluble cell extract, and this allowed us to determine the DNA recognition sequence of the Mtase to be GAAnnnnnnnTTC. This recognition sequence is an inverted repeat of 5'-end of the EcoR1241 recognition sequence. This suggests that the mutant Mtase is assembled from two inverted HsdS(D50) subunits, possibly held together by the HsdM subunits.     

Analysis of type I restriction modification systems in the Neisseriaceae: genetic organization and properties of the gene products

The hsd locus (host specificity of DNA) was identified in the Neisseria gonorrhoeae genome. The DNA fragment encoding this locus produced an active restriction and modification (R/M) system when cloned into Escherichia coli. This R/M system was designated NgoAV. The cloned genomic fragment (7800 bp) has the potential to encode seven open reading frames (ORFs). Several of these ORFs had significant homology with other proteins found in the databases: ORF1, the hsdM, a methylase subunit (HsdM); ORF2, a homologue of dinD; ORF3, a homologue of hsdS; ORF4, a homologue of hsdS; and ORF5, an endonuclease subunit hsdR. The endonuclease and methylase subunits possessed strongest protein sequence homology to the EcoR124II R/M system, indicating that NgoAV belongs to the type IC R/M family. Deletion analysis showed that only ORF3 imparted the sequence specificity of the RM.NgoAV system, which recognizes an interrupted palindrome sequence (GCAN(8-)TGC). The genetic structure of ORF3 (208 amino acids) is almost identical to the structure of the 5' truncated hsdS genes of EcoDXXI or EcoR124II R/M systems obtained by in vitro manipulation. Genomic sequence analysis allowed us to identify hsd loci with a very high homology to RM.NgoAV in two strains of Neisseria meningitidis. However, significant differences in the organization and structure of the hsdS genes in both these systems suggests that, if functional, they would possess recognition sites that differ from the gonococcus and from themselves.     

The EcoR124 and EcoR124/3 restriction and modification systems: Cloning the genes

The Escherichia coli plasmid R124 codes for a type I restriction and modification system EcoR124 and carries genetic information, most probably in the form of a "silent copy," for the expression of a different R-M specificity R124/3. Characteristic DNA rearrangements have been shown to accompany the switch in specificity from R124 to R124/3 and vice versa. We have cloned a 14.2-kb HindIII fragment from R124 and shown that it contains the hsdR, hsdM, and hsdS genes which code for the EcoR124 R-M system. An equivalent fragment from the plasmid R124/3 following the switch in R-M specificity has also been cloned and shown to contain the genes coding for the EcoR124/3 R-M system. These fragments, however, lack a component present on the wild-type plasmid essential for the switch in specificity. Restriction fragment maps and preliminary heteroduplex analysis indicate the near identity of the genes that encode the two different DNA recognition specificities. Transposon mutagenesis was used to locate the positions of the hsdR, hsdM, and hsdS genes on the cloned fragments in conjunction with complementation tests for gene function. Indirect evidence indicates that hsdR is expressed from its own promoter and that hsdM and hsdS are expressed from a single promoter, unidirectionally.     

Cloning and nucleotide sequences of the BanI restriction-modification genes in Bacillus aneurinolyticus

The genes of the BanI restriction-modification system specific for GGPyPuCC were cloned from the chromosomal DNA of Bacillus aneurinolyticus IAM1077, and the coding regions were assigned on the nucleotide sequence on the basis of the N-terminal amino acid sequences and molecular weights of the enzymes. The restriction and modification genes coded for polypeptides with calculated molecular weights of 39,841 and 42,637, respectively. Both the enzymes were coded by the same DNA strand. The restriction gene was located upstream of the methylase gene, separated by 21 bp. The cloned genes were significantly expressed in E. coli cells, so that the respective enzymes could be purified to homogeneity. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration indicated that the catalytically active form of the endonuclease was dimeric and that of the methylase was monomeric. Comparison of the amino acid sequences revealed no significant homology between the endonuclease and methylase, though both enzymes recognize the same target sequence. Sequence comparison with other related enzymes indicated that BanI methylase contains sequences common to cytosine-specific methylases.     

Restriction-modification system with methyl-inhibited base excision and abasic-site cleavage activities

The restriction-modification systems use epigenetic modification to distinguish between self and nonself DNA. A modification enzyme transfers a methyl group to a base in a specific DNA sequence while its cognate restriction enzyme introduces breaks in DNA lacking this methyl group. So far, all the restriction enzymes hydrolyze phosphodiester bonds linking the monomer units of DNA. We recently reported that a restriction enzyme (R.PabI) of the PabI superfamily with half-pipe fold has DNA glycosylase activity that excises an adenine base in the recognition sequence (5′-GTAC). We now found a second activity in this enzyme: at the resulting apurinic/apyrimidinic (AP) (abasic) site (5′-GT#C, # = AP), its AP lyase activity generates an atypical strand break. Although the lyase activity is weak and lacks sequence specificity, its covalent DNA–R.PabI reaction intermediates can be trapped by NaBH₄ reduction. The base excision is not coupled with the strand breakage and yet causes restriction because the restriction enzyme action can impair transformation ability of unmethylated DNA even in the absence of strand breaks in vitro. The base excision of R.PabI is inhibited by methylation of the target adenine base. These findings expand our understanding of genetic and epigenetic processes linking those in prokaryotes and eukaryotes.     

The specificity of sty SKI, a type I restriction enzyme, implies a structure with rotational symmetry.

The type I restriction and modification (R-M) enzyme from Salmonella enterica serovar kaduna ( Sty SKI) recognises the DNA sequence 5'-CGAT(N)7GTTA, an unusual target for a type I R-M system in that it comprises two tetranucleotide components. The amino target recognition domain (TRD) of Sty SKI recognises 5'-CGAT and shows 36% amino acid identity with the carboxy TRD of Eco R124I which recognises the complementary, but degenerate, sequence 5'-RTCG. Current models predict that the amino and carboxy TRDs of the specificity subunit are in inverted orientations within a structure with 2-fold rotational symmetry. The complementary target sequences recognised by the amino TRD of Sty SKI and the carboxy TRD of Eco R124I are consistent with the predicted inverted positions of the TRDs. Amino TRDs of similar amino acid sequence have been shown to recognise the same nucleotide sequence. The similarity reported here, the first example of one between amino and carboxy TRDs, while consistent with a conserved mechanism of target recognition, offers additional flexibility in the evolution of sequence specificity by increasing the potential diversity of DNA targets for a given number of TRDs. Sty SKI identifies the first member of the IB family in Salmonella species.     

Domain movement within a gene: a novel evolutionary mechanism for protein diversification

A protein function is carried out by a specific domain localized at a specific position. In the present study, we report that, within a gene, a specific amino acid sequence can move between a certain position and another position. This was discovered when the sequences of restriction-modification systems within the bacterial species Helicobacter pylori were compared. In the specificity subunit of Type I restriction-modification systems, DNA sequence recognition is mediated by target recognition domain 1 (TRD1) and TRD2. To our surprise, several sequences are shared by TRD1 and TRD2 of genes (alleles) at the same locus (chromosomal location); these domains appear to have moved between the two positions. The gene/protein organization can be represented as x-(TRD1)-y-x-(TRD2)-y, where x and y represent repeat sequences. Movement probably occurs by recombination at these flanking DNA repeats. In accordance with this hypothesis, recombination at these repeats also appears to decrease two TRDs into one TRD or increase these two TRDs to three TRDs (TRD1-TRD2-TRD2) and to allow TRD movement between genes even at different loci. Similar movement of domains between TRD1 and TRD2 was observed for the specificity subunit of a Type IIG restriction enzyme. Similar movement of domain between TRD1 and TRD2 was observed for Type I restriction-modification enzyme specificity genes in two more eubacterial species, Streptococcus pyogenes and Mycoplasma agalactiae. Lateral domain movements within a protein, which we have designated DOMO (domain movement), represent novel routes for the diversification of proteins.     

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.