Type IA enzymes

Type IA restriction endonucleases

Acknowledgement: Much of the material below is taken directly from the abstracts of published papers and as a consequence I have cited the relevant paper in each section. My thanks to all those workers who have contributed either knowingly or unknowingly to this page (KF).

R.EcoKI is a member of the Type I IA group (K-family) of restriction and modification (R-M) enzymes (for recent reviews see Bickle and Kruger (1987) and Wilson and Murray (1991). These multifunctional enzymes are able to both cleave and methylate double-stranded DNA, and are potentially important models for studying both protein-DNA and protein-protein interactions. The gene order is hsdR, M, S, the hsdR gene has its own promoter P_RES and the hsdM and hsdS genes are transcribed from a single promoter (P_MOD) situated between genes hsdR and hsdM.

The DNA target sequences recognised by Type I R-M enzymes consist of two specific components, one of 3-bp and another of 4- or 5-bp, separated by a non-specific spacer,

e. g.

AACN₆GTGC for EcoKI

TGAN₈TGCT for EcoBI.

Sequence comparisons between the hsdS genes of the EcoKI, EcoBI and EcoDI systems revealed two extensive variable regions of about 450-bp in length and two other regions, 100-bp in the centre and 250-bp at the distal end, which show high degrees of conservation. It was suggested that the conserved regions are involved in protein-protein interactions and the variable regions in DNA recognition. Recent studies have shown that the proximal variable region specifies recognition of the trinucleotide component of the DNA target sequence. Two members of the A-family of R-M systems, EcoAI and EcoEI, recognise GAG and show 80% identity of their proximal variable regions, however 44% identity is sufficient for recognition as shown by comparison of the sequences of the hsdS genes of EcoAI or EcoEI with SB from K the family which also recognises the trinucleotide GAG. DNA recognition specificity lies not in the entire variable region but only in parts of this region.

DNA cleavage

The DNA intermediates and final products formed by the Type IA restriction endonuclease, EcoBI, were described by Endlich and Linn (1985b). DNA cleaved on only one strand (hemi-restricted DNA) contains gaps of approximately 70-100 nucleotides, while the fully restricted products contain 3'-single-stranded tails averaging approximately 70-100 nucleotides for each strand cleaved. The gaps and tails are formed with the release of an equal number of nucleotides as small oligonucleotides that are soluble in acid. After purification, neither the hemi-restricted nor the fully restricted DNAs are cleaved again by EcoB. There is no apparent specificity for which strand of a duplex is initially cleaved by EcoB, nor is there specificity with respect to the composition of the 3'-terminal nucleotide formed on the DNA - or the 3'- or 5'-terminal nucleotides of the acid soluble oligonucleotides released during DNA cleavage. The structure formed at the 5' terminus of the DNA product which blocks phosphorylation by T4 polynucleotide kinase remains unknown, but its removal with phage l exonuclease allows at least some reutilization of recognition sites by EcoB as well as phosphorylation of the newly formed 5' termini. To explain the complex mechanism of this enzyme, it is suggested that the unidentified 5'-tails prevent wasteful restriction from occurring, whereas the 3'-single. stranded tails create DNA which, when nonhomologous to chromosomal DNA, cannot be rescued because such tails are not substrate for DNA polymerases. However, when homologous chromosomal DNA exists, the randomly cleaved large fragments with these tails can easily be assimilated by recA-mediated genetic recombination, thus stimulating DNA exchange between related organisms.

DNA binding

Cooper and Dryden (1994) describe the two DNA binding domains on the DNA specificity (S) subunit and amino acid motifs for binding the methyl group donor, S-adenosyl methionine (AdoMet) on the other two modification (M) subunits. They show that type Ia family of enzymes, including the E. coli K12 system, respond differently to DNA targets which have one of two adenine bases in the target methylated at the N6 position, than to those targets which have neither adenine methylated. The hemi-methylated targets are the strongly preferred substrates for methylation. They have used partial proteolytic digestion of the E. coli K12 methyltransferase, EcoKI, to generate polypeptide domains which have been identified by amino acid sequencing. The subunit was cut into two large fragments at the site near to the small region connecting the predicted DNA binding domains. One of these fragments was sufficiently stable to remain associated with a fragment of the M subunit. By comparison with the proposed structure of S subunits this contact with the M subunit involves the region between the two DNA binding domains. Binding of DNA but not of AdoMet partially protected the S subunit from digestion. the M subunit was also cut into two domains at one of the AdoMet binding motifs. This motif would appear to be on an exposed loop and the binding of AdoMet protected the loop from proteolysis. the second domain remained associated with the fragment of the S subunit apparently contacting the region of S linking the two DNA binding domains. The M subunit also has a proteolytically sensitive tail, the conformation of which is affected by the formation of a ternary complex with AdoMet and DNA but not with AdoMet or DNA alone. In the ternary complex, the tail region is digested differently depending on whether the DNA is unmethylated or hemimethylated. This suggests that this region is involved in the recognition of DNA methylation.

The EcoKI methyltransferase methylates two adenines on opposite strands of its bipartite DNA recognition sequence AACN₆GTGC. The enzyme has a strong preference for hemimethylated DNA substrates, but the methylation state of the DNA does not influence its binding affinity. Powell and Murray (1995) used methylation interference to compare contacts made by the EcoKI methyltransferase with unmodified, hemimethylated or fully modified DNAs. Contacts were seen at or near the N7 position of guanine, in the major groove, for all of the guanines in the EcoKI recognition sequence, and at two guanines on the edge of the intervening spacer sequence. The presence of the cofactor and methyl donor S-adenosyl methionine had a striking effect on the interference pattern for unmodified DNA which could not be mimicked by the presence of the cofactor analogue S-adenosyl homocysteine. In contrast, S-adenosyl methionine had no effect on the interference patterns for either kind of hemimethylated DNA, or for fully modified DNA. Differences between the interference patterns for the unmodified DNA and any of three forms of methylated DNA provide evidence that methylation of the target sequence influences the conformation of the protein-DNA interface, and illustrate the importance of S-adenosyl methionine in the distinction between unmodified and methylated DNA by the methyltransferase.

Chen et al. (1995) describe the identification of an important amino acid involved in DNA binding using cross-linking to DNA. The specificity (S) subunit of the restriction enzyme EcoKI imparts specificity for the sequence AACN₆GTGC. Substitution of thymine with bromodeoxyuridine in a 25 bp DNA duplex containing this sequence stimulated UV light-induced covalent crosslinking to the S subunit. Crosslinking occurred only at the residue complementary to the first adenine in the AAC sequence, demonstrating a close contact between the major groove at this sequence and the S subunit. peptide sequencing of a proteolytically-digested, crosslinked complex identified tyrosine 27 in the S subunit as the site of crosslinking. This is consistent with the role of the N-terminal domain of the S subunit in recognizing the AAC sequence. Tyrosine 27 is conserved in the S subunits of the three Type I enzymes that share the sequence AA in the trinucleotide component of their target sequence. This suggests that tyrosine 27 may make a similar DNA contact in these other enzymes.

The Type I restriction and modification enzymes do not possess obvious DNA-binding motifs within their target recognition domains (TRDs) of 150-180 amino acids. To identify residues involved in DNA recognition, O'Neill et al. (1998) made changes were made in the amino-TRD of EcoKI by random mutagenesis. Most of the 101 substitutions affecting 79 residues had no effect on the phenotype. Changes at only seven positions caused the loss of restriction and modification activities. The seven residues identified by mutation are not randomly distributed throughout the primary sequence of the TRD: five are within the interval between residues 80 and 110. Sequence analyses have led to the suggestion that the TRDs of Type I restriction enzymes include a tertiary structure similar to the TRD of the HhaI methyltransferase, and to a model for a DNA-protein interface in EcoKI. In this model, the residues within the interval identified by the five mutations are close to the protein-DNA interface. Three additional residues close to the DNA in the model were changed; each substitution impaired both activities. Proteins from twelve mutants were purified: six from mutants with partial or wild-type activity and six from mutants lacking activity. There is a strong correlation between phenotype and DNA-binding affinity, as determined by fluorescence anisotropy.

The Type I DNA restriction and modification enzymes of prokaryotes are multimeric enzymes that cleave unmethylated, foreign DNA in a complex process involving recognition of the methylation status of a DNA target sequence, extensive translocation of DNA in both directions towards the enzyme bound at the target sequence, ATP hydrolysis, which is believed to drive the translocation possibly via a helicase mechanism, and eventual endonucleolytic cleavage of the DNA. Powell et al. (1998) have examined the DNA binding affinity and exonuclease III footprint of the EcoKI type IA restriction enzyme on oligonucleotide duplexes that either contain or lack the target sequence. The influence of the cofactors, S-adenosyl methionine and ATP, on binding to DNA of different methylation states has been assessed. EcoKI in the absence of ATP, with or without S-adenosyl methionine, binds tightly even to DNA lacking the target site and the exonuclease footprint is large, approximately 45 base-pairs. The protection is weaker on DNA lacking the target site. Partially assembled EcoKI lacking one or both of the subunits essential for DNA cleavage, is unable to bind tightly to DNA lacking the target site but can bind tightly to the recognition site. The addition of ATP to EcoKI, in the presence of AdoMet, allows tight binding only to the target site and the footprint shrinks to 30 base-pairs, almost identical to that of the modification enzyme which makes up the core of EcoKI. The same effect occurs when S-adenosyl homocysteine or sinefungin are substituted for S-adenosyl methionine, and ADP or ATPgS are substituted for ATP. It is proposed that the DNA binding surface of EcoKI comprises three regions: a "core" region which recognises the target sequence and which is present on the modification enzyme, and a region on each DNA cleavage subunit. The cleavage subunits make tight contacts to any DNA molecule in the absence of cofactors, but this contact is weakened in the presence of cofactors to allow the protein conformational changes required for DNA translocation when a target site is recognised by the core modification enzyme. This weakening of the interaction between the DNA cleavage subunits and the DNA could allow more access of exonuclease III to the DNA and account for the shorter footprint.

DEAD box motifs

One subunit of both Type I and Type III restriction and modification enzymes contains motifs characteristic of DEAD box proteins, which implies that these enzymes may be DNA helicases. This subunit is essential for restriction, but not modification. The current model for restriction by both types of enzyme postulates that DNA cutting is stimulated when two enzyme complexes bound to neighbouring target sequences meet as the consequence of ATP-dependent DNA translocation. For Type I enzymes, this model is supported by in vitro experiments, but the predicted co-operative inter-actions between targets have not been detected by assays that monitor restriction in vivo. The experiments reported by Webb et al. (1996) clearly establish the required synergistic effect but, in contrast to earlier experiments, they use Escherichia coli K-12 strains deficient in the restriction alleviation function associated with the Rac prophage. In bacteria with elevated levels of EcoKI the cooperative interactions are obscured, consistent with co-operation between free enzyme and that bound at target sites. We have made changes in three of the motifs characteristic of DEAD box proteins, including motif III, which in RecG is implicated in the migration of Holliday junctions. Conservative changes in each of the three motifs impair restriction.

For Type I restriction systems, recently determined nucleotide sequences predict conserved amino acids in the subunit that is essential for restriction but not modification (HsdR). The conserved sequences emphasize motifs characteristic of the DEAD-box family of proteins which comprises putative helicases, and they identify a new candidate for motif IV. Davies et al. (1998) provide evidence based on an analysis of EcoKI which supports both the relevance of DEAD-box motifs to the mechanism of restriction and the new definition of motif IV. Amino acid substitutions within the newly identified motif IV and those in six other previously identified DEAD-box motifs, but not in the original motif IV, confer restriction-deficient phenotypes. We have examined the relevance of the DEAD-box motifs to the restriction pathway by determining the steps permitted in vitro by the defective enzymes resulting from amino acid substitutions in each of the seven motifs. EcoKI purified from the seven restriction-deficient mutants binds to an unmethylated target sequence and, in the presence of AdoMet, responds to ATP by undergoing the conformational change essential for the pathway of events leading to DNA cleavage. The seven enzymes have little or no ATPase activity and no endonuclease activity, but they retain the ability to nick unmodified DNA, though at reduced rates. Nicking of a DNA strand could therefore be an essential early step in the restriction pathway, facilitating the ATP-dependent translocation of DNA, particularly if this involves DNA helicase activity.

Subunit assembly

It has been noted that the genes specifying Type I systems can be transferred to a new host lacking the appropriate, protective methylation without any adverse effect. Therefore, this situation has been analysed by Dryden et al. (1997). The modification phenotype apparently appears before the restriction phenotype, but no evidence for transcriptional or translational control of the genes and the resultant phenotypes has been found. Type I I enzymes contain three types of subunit, S for sequence recognition, M for DNA modification (methylation), and R for DNA restriction (cleavage), and can function solely as a M₂S₁ methylase or as a R₂M₂S₁ bifunctional methylase/nuclease. We show that the methylase is not stable at the concentrations expected to exist in vivo, dissociating into free M subunit and M₁S₁, whereas the complete nuclease is a stable structure. The M₁S₁ form can bind the R subunit as effectively as the M₂S₁ methylase but possesses no activity; therefore, upon establishment of the system in a new host, we propose that most of the R subunit will initially be trapped in an inactive complex until the methylase has been able to modify and protect the host chromosome. We believe that the in vitro assembly pathway will reflect the in vivo situation, thus allowing the assembly process to at least partially explain the observations that the modification phenotype appears before the restriction phenotype upon establishment of a Type I system in a new host cell.

The Type I DNA restriction and modification systems of enteric bacteria display several enzymatic activities due to their oligomeric structure. Partially assembled forms of the EcoKI enzyme from E. coli K12 can display specific DNA binding properties and modification methyltransferase activity. The heterodimer of one specificity (S) subunit and one modification (M) subunit can only bind DNA whereas the addition of a second modification subunit to form M₂S₁ also confers methyltransferase activity. Powell et al. (1998) have examined the DNA binding specificity of M₁S₁ and M₂S₁ using the change in fluorescence anisotropy which occurs on binding of a DNA probe labelled with a hexachlorofluorescein fluorophore. The dimer has much weaker affinity for the EcoKI target sequence than the trimer and slightly less ability to discriminate against other DNA sequences. Binding of both proteins is strongly dependent on salt concentration. The fluorescence results compare favourably with those obtained with the gel retardation method. DNA footprinting using exonuclease III and DNase I, and methylation interference show no asymmetry, with both DNA strands being protected by the dimer and the trimer. This indicates that the dimer is a mixture of the two possible forms, M₁S₁ and S₁M₁. The dimer has a footprint on the DNA substrate of the same length as the trimer implying that the modification subunits are located on either side of the DNA helical axis rather than lying along the helical axis.

Temperature-sensitive mutations

The genes hsdM and hsdS for M.EcoKI modification methyltransferase and the complete set of hsdR, hsdM and hsdS genes encoding for R.EcoKI restriction endonuclease, both with and without a temperature-sensitive (ts) mutation in hsdS gene, were cloned by Weiserova et al. (1994) in pBR322 plasmid and introduced into E. coli C (a strain without a natural restriction-modification (R-M) system). the strains producing only methyltransferase, or together with endonuclease, were thus obtained. The hsdSts-1 mutation, mapped previously in the distal variable region of the hsdS gene with C¹²⁴⁵-T transition has no effect on the R-M phenotype expressed from cloned genes in bacteria grown at 42°C. In clones transformed with the whole hsd region an alleviation of R-M functions was observed immediately after transformation, but after subculture the transformants expressed the wild-type R-M phenotype irrespective of whether the wild-type or mutant hsdS allele was present in the hybrid plasmid. Simultaneous overproduction of HsdS and HsdM subunits impairs the ts effect of the hsdSts-1 mutation on restriction and modification.

Further analysis of the above mutation by Janscak et al. (2000) and another mutation in the hsdS gene designated Sts2 (Ala²⁰⁴Thr) show that they had a different impact on restriction-modification functions in vitro and in vivo. The enzyme activities of the Sts1 mutant were temperature sensitive in vitro and were reduced even at 30°C (permissive temperature). Gel retardation assays revealed that the Sts1 mutant had significantly decreased DNA binding, which was temperature-sensitive. In contrast the Sts2 mutant did not show differences from the wild-type enzyme even at 42°C. Unlike the HsdS_ts1 subunit, the HsdSts2 subunit was not able to compete with the wild-type subunit in assembly of the restriction enzyme in vivo, suggesting that the Sts2 mutation affects subunit assembly. Thus, it appears that these two mutations map two important regions in HsdS subunit responsible for DNA-protein and protein-protein interactions, respectively. Interestingly the Sts2 mutation maps to region of the subunit that has been shown to be involved in the production of non-classical phenotypes for the type IC R-M system EcoR124I suggesting this region is very important for protein-protein interactions (Weiserova & Firman, 1998).

Control of restriction-modification functions

Efficient acquisition of genes that encode a restriction and modification (R-M) system with specificities different from any already present in the recipient bacterium requires the sequential production of the new modification enzyme followed by the restriction activity in order that the chromosome of the recipient bacterium is protected against attack by the restriction endonuclease. Makovets et al. (1998) show that ClpX and ClpP, the components of ClpXP protease, are necessary for the efficient transmission of the genes encoding EcoKI and EcoAI, representatives of two families of Type I R-M systems, thus implicating ClpXP in the modulation of restriction activity. Loss of ClpX imposed a bigger barrier than loss of ClpP, consistent with a dual role for ClpX, possibly as a chaperone and as a component of the ClpXP protease. Transmission of genes specifying EcoKI was more dependent on ClpX and ClpP than transmission of the genes for EcoAl. Sensitivity to absence of the protease was also influenced by the mode of gene transfer; conjugative transfer and transformation were more dependent on ClpXP than transduction. In the absence of either ClpX or ClpP transfer of the EcoKI genes by P1-mediated transduction was impaired, transfer of the EcoAl genes was not.

ClpXP-dependent proteolysis has been implicated in the delayed detection of restriction activity after the acquisition of the genes (hsdR, hsdM, and hsdS) is that specify EcoKI and EcoAI, representatives of two families of Type I restriction and modification (R-M) systems. Modification, once established, has been assumed to provide adequate protection against a resident restriction system. However unmodified targets may be generated in the DNA of an hsd⁺ bacterium as result of replication errors or recombination-dependent repair. Makovets et al. (1999) show that ClpXP-dependent regulation of the endonuclease activity enables bacteria the choir on mortified chromosomal target sequences to survive. In such bacteria, HsdR, the polypeptide of R-M complex essential for restriction but not modification, is degraded in the presence of ClpXP. A mutation that blocks only the modification activity of EcoKI, leaving the cell with approximately 600 unmodified targets, is not lethal provided that ClpXP is present. Their data support a model in which the HsdR component of a Type I restriction endonuclease becomes a substrate for proteolysis after the endonuclease has bound to unmodified target sequences, but before completion of the pathway that would result in DNA breakage.

Makovets et al observed that despite the proteolysis of the HsdR that comprises unmodified DNA-bound endonuclease, some restriction activity remained in the cell. We have suggested that this activity may result from some membrane bound endonuclease (Holubova et al., 2000).

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