Biology of DNA restriction

Our understanding of the evolution of DNA restriction and modification systems, the control of the expression of the structural genes for the enzymes, and the importance of DNA restriction in the cellular economy has advanced by leaps and bounds in recent years. This review documents these advances for the three major classes of classical restriction and modification systems, describes the discovery of a new class of restriction systems that specifically cut DNA carrying the modification signature of foreign cells, and deals with the mechanisms developed by phages to avoid the restriction systems of their hosts.


INTRODUCTION
The phenomenon of restriction and modification (R-M) of bacterial viruses was first described some 40 years ago (15,125) and received a molecular explanation 10 years later: restriction was due to an endonucleolytic cleavage of foreign DNA (2,3,51). We now know that there are two different types of restriction systems: those in which cellular DNA is protected from restriction by modification methylation of adenosyl or cytosyl residues within the sequences recognized by the restriction enzymes (the classical R-M systems) and those which cleave only DNA that carries specific modifications. A large body of knowledge concerning DNA restriction has accumulated and has been the subject of many excellent reviews (5, 18, 19, 24, 57, 100, 102, 120, 124, 131-133, 157, 168, 169, 214, 219).
The purpose of this review is to describe the exciting developments in our understanding of the biology of DNA restriction during the last 10 years. We will begin by discussing recent findings with the classical type I, II, and III R-M systems. We will continue with the rediscovery of a group of restriction systems that specifically cleave DNA carrying certain modifications; host cell DNA in this case avoids restriction because it is not modified. Finally, we will discuss the ways in which bacteriophages avoid the effects of the R-M systems of their hosts. The major characteristics of the different kinds of R-M systems are set out in Table 1.

TYPE I R-M SYSTEMS
The type I R-M systems are the most complex so far discovered. The main enzyme is a three-subunit protein containing the products of the hsdS, hsdM, and hsdR genes (hsd for host specificity for DNA). This enzyme is multifunctional, catalyzing both restriction and modification reactions, and is also a restriction-dependent ATPase and a DNA topoisomerase (reviewed in reference 19). A second enzyme containing the products of the hsdS and hsdM genes can also be isolated. This enzyme is a monofunctional modification methylase, which may have no physiological role under normal conditions since the three-subunit enzyme is also an efficient modification methylase. Type I systems have so far only been found in members of the family Enterobacteriaceae, with the exception of an early report of a member in Haemophilus influenzae (65), which may actually be a type Cut DNA with modified C and A tion enzymes residues III system (the distinction between types I and III was first made in 1978 [88]).

Type I Systems Form Families of Related Enzymes
The major finding of the last few years has been that the enterobacterial type I systems are grouped in genetically related families, a fact that has important consequences for the understanding of the biology of these systems. Early studies had showed that many of the chromosomally coded type I R-M systems could genetically complement each other; indeed, complementation was the tool that allowed the number and function of the hsd genes to be defined (25). By chance, all of the R-M systems from Escherichia coli and Salmonella spp. that were investigated in the early days were from the same family and thus gave genetic complementation. The first hint that different families might exist came from a comparison of genes for different systems by DNA hybridization and from an immunochemical comparison of different enzymes by using hybridization probes and antisera specific for the EcoKI enzyme of E. coli K-12 (135). It was found that for all except one of the systems investigated there was considerable homology among the structural genes and that two of the three subunits of the enzymes were immunologically cross-reactive (no antisera were available for the third subunit). It was proposed that EcoAI, the exceptional nonhomologous enzyme, should be considered the first member of a new family of type I R-M systems (135).
What are the reasons for considering EcoAI to be a type I system? First, it is allelic to the other type I systems in the sense that the structural genes have the same chromosomal location (6,40); second, the enzymes have a typical type I subunit structure, genetic organization, cofactor requirements, reaction mechanism, and recognition sequence (59,190,192). Finally, EcoAI is the first member of a new family, since two other members were identified later: EcoEI and CfrII (40,59).
A third family of type I systems that has no homology with the other two families and which will not complement them has since been found (63,150,155,179). The first member of this family to be investigated was StyR124I, coded by a Salmonella conjugative plasmid. Most members of the family are plasmid coded (for a possibly trivial exception, see reference 119), and thus they cannot be said to be allelic to the other two families. However, in all other features they are typical type I systems. It has been proposed that the three type I families be called type IA with EcoKI as the prototype, type IB (the EcoAI family), and type IC (StyR124I and relatives) (19). All of the characterized type I R-M systems, together with their recognition sequences, are shown in Table 2.

Structure of hsd Genes
The structural genes for all three families of type I systems have now been characterized at the molecular level by DNA sequencing or DNA hybridization analysis (38,40,59,60,62,64,67,86,119,122,135,154,174). Several generalities emerge from a comparison of these sequences. First, all of the genes are arranged into two contiguous transcription units, with hsdM and hsdS forming an operon and hsdR being transcribed from its own promoter; the order of the two transcriptional units is different for different families (Fig. 1). Second, with one important exception (see below), there is no sequence homology between different families, apart from a few short amino acid sequence motifs common to DNA adenine methylases and to ATP-binding proteins. Third, within a given family the hsdM and hsdR genes are largely homologous throughout their length. Fourth, the hsdS genes contain two regions of high homology (three for the type IB family [86]) separating two extensive regions of nonhomology. There is good evidence (see the next section) for all three families that the regions of nonhomology code two protein domains, each of which recognizes one-half of the split recognition sequences that are characteristic of type I R-M systems ( Table 2). The conserved regions of the hsdS genes are thought to be responsible for protein-protein interactions with the hsdM and hsdR gene products, although there is no hard evidence for this. For the type IC family (see below), there is evidence that the central conserved region of the hsdS gene products also determines, in part, the DNA recognition properties of the enzymes.
Evolution of DNA Sequence Recognition by Recombination between hsdS Genes Type I R-M systems are the only examples known to date of proteins that recognize specific DNA sequences and that can change their sequence specificities by well-understood natural processes. The first of these to be discovered was in the type IA family, when an attempt to transduce the structural genes for StySPI into cells containing the StySBI genes resulted in a new specificity, which was called StySQI (30). It was then shown, first by electron-microscopic heteroduplex analysis and later by DNA sequencing, that StySQI was the result of a recombination event within the central conserved region of the hsdS genes of StySPI and StySBI (58,60). The DNA sequences recognized by the two parents and by the recombinant are shown in Table 2 (136,137). It is clear that the StySQI recognition sequence is a hybrid between those of the two parents: the 5' end of the sequence is the same as the 5' half of the StySPI sequence, and the 3' end of the sequence is identical to that of the 3' half of the StySBI sequence.
The picture that emerges from these results is that the recombination event that created the StySQI hsdS gene reassorted two protein domains, each of which recognizes one-half of the split recognition sequence. This idea is strengthened by the following results. If the model is correct, the reciprocal recombinant, that is, with 5' sequences from the StySBI hsdS gene and 3' sequences from the StySPI hsdS gene, should have a predictable hybrid recognition sequence. This recombinant was made and proved to have the predicted recognition sequence (StySJI in Table 2) (62). Strong support for the hypothesis also comes from studies with the type IB enzyme EcoEI (Table 2). This enzyme recognizes GAG as the 5' part of its recognition sequence, as do EcoAI itself and the type IA enzyme StySBI (  (63). The DNA sequences recognized by the two systems are shown in Table 2. They differ only in the length of the nonspecific spacer separating the two half-sites of the sequence: StyR124I requires a 6-bp spacer, whereas StyR124/3I requires a 7-bp spacer. In terms of enzymatic recognition of DNA, this is not a trivial difference. The extra base pair in the StyR124/3I spacer moves the two half-sites 3.4 A (0.34 nm) further apart and rotates them by 360 with respect to the StyR124I sequence. The difference between the two enzymes was determined by sequencing the structural genes. The two sequences were identical except at one position in the middle of the hsdS genes, where a 12-bp in-frame sequence was directly repeated twice in the StyR124I gene and three times in the StyR124/31 gene (154).
The mechanism of the switch in specificity was immediately clear: unequal crossing over between the two repeats of the StyR124I hsdS gene would generate one copy of the gene with three repeats and a second copy with one repeat. A similar mechanism could reduce the number of repeats from three back to two again. This result led to the hypothesis that the region of the protein that contains the repeated sequences forms a spacer separating two DNA-binding domains, each of which binds to one-half of the recognition sequence. The extra four amino acids in the spacer of the StyR124/3I protein would increase the physical distance between the DNA-binding domains sufficiently (6 A [0.6 nm] if a-helical) that they could no longer bind a recognition sequence with 6 bp separating the two halves but could make productive interactions with a sequence containing a 7-bp spacer.
This hypothesis gained support from results of mutagenesis experiments examining the region of the repeats (66). Single-amino-acid replacements within the repeats had no effect on either activity or specificity, indicating that the exact amino acid sequence in this region of the protein is not crucial for its function. Changes in the length of the repeated region had profound effects on both activity and specificity. For example, variants with one or four copies of the repeated sequences were very inefficient restriction enzymes, 10-5 to 10-6 times less effective than the wild-type enzymes, which explains why these variants were not found naturally. They were, on the other hand, efficient modification enzymes: the variant with one repeat methylated the StyR124I sequence, and the enzyme with four repeats methylated both StyR124I and StyR124/3I sequences, as though its long protein spacer was flexible enough to be floppy. These differences between restriction and modification are interesting and may indicate that the region of the spacer serves a double function, determining the length of the nonspecific spacer in the recognition sequence on the one hand and mediating protein-protein interactions between HsdS and HsdR on the other.
Another enzyme in the type IC family is EcoDXXI, which recognizes a DNA sequence completely different from that recognized by StyR124I or StyR124/31 (Table 2). DNA heteroduplex analysis of the structural genes of EcoDXXI and StyR124I showed that the overall structure was the same as in the type IA family: homology throughout the region except within the hsdS genes, where two long blocks of nonhomology were found. A limited DNA sequence analysis of the central region of the hsdS gene showed that EcoDXXI was very similar to StyR124/31; in particular, the three copies of the 12-bp repeated sequence were identical (67). All possible hybrids between the StyR124I and EcoDXXI hsdS genes were constructed with one-half of each hybrid derived from one system, one-half from the other system, and either two or three copies of the 12-bp repeat in the middle. All of these hybrids were active in both restriction and modification, and the sequences that they recognized were determined. The results clearly demonstrate the correlation between protein structure and target site specificity. As with the type IA systems, the amino terminus of the protein recognizes the trinucleotide half of the recognition sequence, the central part of the protein determines the number of nucleotides in the nonspecific spacer, and the carboxyl terminus recognizes the other half of the recognition sequence ( Table 2) (67).

Mutations Affecting Modification Activity
A crucial difference between eukaryotic and all prokaryotic DNA methyltransferases but one concerns the DNA substrate. The mammalian enzymes methylate the cytosine in mainly CG sequences to 5-methylcytosine (5-meC), but they do it efficiently only if the cytosine in the opposite strand already bears a methyl residue. The result is that CG sequences that are methylated perpetuate their methylated state following DNA replication whereas nonmethylated CG sequences normally remain nonmethylated. Methylation is correlated with gene inactivation, and it is thought that DNA methylation is one of the primary control processes in development: as yet undetected enzymes would inactivate a gene by de novo methylation of CG sequences, and the maintenance methylases, which require a hemimethylated substrate, would then ensure that the gene remained inactivated during further cell divisions (reviewed in reference 170). Most prokaryotic DNA methylases behave quite differently. They show no marked preference for hemimethylated over nonmethylated DNA and therefore have the properties expected for the postulated de novo eukaryotic enzyme. The type IA enzymes are an exception to this rule in that both the modification enzymes and the multifunctional restriction enzymes are bona fide maintenance methylases, methylating hemimethylated substrates much more efficiently than they methylate nonmethylated ones (31,191,205). Mutations in the type IA system EcoK that give it the properties expected of a de novo methylase have now been selected (90). The mutations are all loosely clustered within the hsdM gene, indicating that it is the HsdM polypeptide that senses the methylation state of the recognition site. Thus, elements of both HsdS and HsdM must interact directly with the recognition site, and it will be interesting to determine whether these interactions are simultaneous or sequential. Some of these mutations are also defective in restriction, but the molecular basis for the deficiency is not yet known (90).

TYPE II R-M SYSTEMS
The type II systems are the simplest of the R-M systems, with separate restriction and modification enymes and no special cofactor requirements other than Mg"+ ( Table 1).
The fact that the restriction enzymes recognize and cleave simple sequences in DNA at fixed positions has made them indispensable in many if not most branches of molecular biology. These useful properties of type II enzymes have stimulated many laboratories to screen thousands of taxonomically diverse bacteria for enzymes with new specificities. As a result, we know of hundreds of enzymes, most of which, apart from their DNA recognition properties, have not been further characterized.

Evolutionary Aspects
So far, the structural genes from over 50 type II R-M systems have been sequenced (213). Surprisingly, no significant amino acid sequence homologies could be found between cognate pairs of endonucleases and methyltransferases. Although they recognize the same DNA sequence, even their target recognition domains (TRDs) are different (35,114). Therefore, for type II enzymes, the endonucleases and methyltransferases are the products of independent evolution. One can envisage a scenario in which the methyltransferases first evolved primarily as enzymes, like the dam methylase of E. coli (reviewed in reference 12), with important functions in the regulation of gene expression, DNA repair, or replication, and then provided a genetic background with a methylated genome in which restriction endonucleases could evolve.
Not only do endonucleases have no homology with their cognate methyltransferases, but also there are no (with a few exceptions) homologies between different type II restriction endonucleases (35,114). Even isoschizomers, that is, endonucleases from different bacterial species that recognize the same sequence and cut it at the same position, such as BsuRI and NgoPII, are very different (92,189). It can be concluded that endonucleases have not evolved from a single primitive precursor and have acquired different sequence specificities by mutating the TRD. They must be of different evolutionary origin. The few cases in which more or less marked homology between isoschizomeric enzymes has been described are the enzyme pairs EcoRI and RsrI (185), BsuBI and PstI (217), BsuFI and MspI (87), TthHB8I and TaqI (11), FnuDI and NgoPII, and Cfr9I and XmaI (214). However, it should be pointed out that EcoRI is coded by a conjugative plasmid (218) and that the EcoRI-RsrI pair may therefore be the result of horizontal gene transfer.
In contrast to the endonucleases, methyltransferases share many amino acid sequence similarities. This is especially true for the family of 5-meC transferases, which includes one eukaryotic enzyme (16,112,114,152). Several groups have described a common building plan for these enzymes, with a pattern of highly conserved amino acid sequence motifs alternating with variable sequence elements (94,114,152). The TRD is part of the largest variable region, which is always located close to the center of the primary sequence (94,114,207,211).
Studies of the 5-meC methyltransferases coded by the Bacillus phages SP1, SPR, 43T, plls, and H2, which methylate several different DNA sequences, have contributed much to our understanding of the functional domains in these enzymes. Each DNA sequence is recognized by a different TRD. These TRDs have been identified, and the feasibility of engineering new combinations to create enzymes with novel specificities has been demonstrated (9,13,111,201,202,211). In addition to functional TDRs, the phages H2 and pll. have silent TRDs in their methyltransferases which can be experimentally activated by mutation (110). It is not understood how such silent domains are maintained, how multispecific methyltransferases originated evolutionarily, or whether modules, perhaps even complete methyltransferase genes, can be exchanged by horizontal gene transfer between phage and cellular genomes (exchange of complete genes between phages has been demonstrated [143,198]). It is thought that it is the presence of restriction systems in their hosts that selects for the methyltransferase genes and drives their evolution (110), even though not all of the required restriction specificities have so far been demonstrated in Bacillus strains.
Homologous segments are also found in the family of the N6meA methyltransferases, and consensus motifs have been defined. In general, the homologies are less extensive than for the 5-meC methylases and the degree of conservation is not as high (35,68,112,113,139,182). The N4meC methyltransferases are (with the possible exception of MvaI) more similar to the N6meA methyltransferases than to the 5-meC methyltransferases (27,95,197). This is perhaps not surprising since methylation of an exocyclic amino group (the N4 of cytosine or the N6 of adenosine) is mechanistically very different from the methylation of a ring carbon to 5-meC. Lauster (112) has developed a model for the evolution of type II methyltransferases by gene duplication and subsequent divergent evolution of the duplicated segments.

Control of Expression of Type II RM Genes
The expression of genes coding for methyltransferases and endonucleases must be tightly regulated. It is essential that the methyltransferase completely modify the cellular genome at all times to protect it from the lethal action of the endonuclease. This regulation is especially important when R-M genes first enter a cell, on conjugative plasmids for example, but it can also be important during changes in the physiology of the cell (e.g., entry into the stationary phase, starvation associated with a deficiency of methyl donors, and other stresses).
For at least some type II R-M systems, this regulation seems to be achieved by the product of an additional open reading frame (ORF) tightly linked to the R-M gene cluster. Tao et al. (195,196) have sequenced this ORF in the PvuII gene cluster and shown that its mutation leads to a restrictionless phenotype even though the structural gene for the endonuclease is intact. The defect can be complemented by a wild-type copy of the ORF in trans. They have proposed that the proteins coded by these ORFs should be known as C proteins (e.g., PvuIIC), where C stands for controller. Brooks and coworkers (27,140) have similarly shown that an ORF in the BamHI system has regulatory functions. Disruption of the BamHIC gene leads to both a decrease in restriction and an increase in modification methylase activity (82). The predicted amino acid sequences of these C proteins have some homology with DNA-binding proteins of the helix-turn-helix class (72), indicating that they may function as transcriptional repressors or activators.
Of the type II R-M systems for which the gene organization and sequence are known, nine systems have an ORF in addition to the R and M structural genes (213). Five of these systems were compared with each other, and for four of them (BamHI, EcoRV, PvuII, and SmaI) the ORFs were remarkably similar, especially in the region of the putative helix-turn-helix motif (195,196). Since there is no homology between the corresponding four endonucleases and the homology between the four methyltransferases (all being either N4meC or N6meA methyltransferases) is also lower than for the C proteins, Tao et al. (196) concluded that the evolution of the C gene family could have been independent of the R and M genes. It is interesting that the BsuRI ORF that has no homology with the other four is from an R-M system that specifies 5-meC (196). Other possible regulatory mechanisms for the expression of type II R-M systems have been proposed (75,180,199,212).
One problem with studies on regulation is that they are almost always done with cloned genes examined in heterologous hosts, in which regulation need not necessarily be the same as in the original strain. Although this was probably not critical for the study with the PvuII gene cluster in E. coli, because PvuII originally comes from Proteus vulgaris, which is also a member of the Enterobacteriacae, it was definitely a factor in the work with the BamHI genes. BamHI comes from the gram-positive sporeformer Bacillus amyloliquifaciens, a close relative ofB. subtilis. It was found that the regulation in the gram-negative E. coli was distinctly different from that in B. subtilis (27).
Cytosine Can Be Methylated on Either C-5 Or N4: Consequences for Mutagenesis All the early examples of DNA modification that had been studied were due either to methylation of the extracyclic amino group of adenosine to N6meA or to methylation of a ring carbon of cytosine to 5-meC. In 1983 Janulaitis et al. discovered that the BcnI methylase from Bacillus centrosporus methylated the extracyclic amino group of cytosine to N4meC (84). N4meC has since been shown to be the product of many modification methylases (32,33,54,56).
It was proposed that N4meC rather than 5-meC was an adaptation to thermophilia, which would avoid the hypermutability associated with 5-meC (54). Deamination of 5-meC is a highly temperature-dependent reaction which produces thymine, resulting in G. C-to-A . T transition mutations on DNA replication. N4meC (as well as C) also undergoes spontaneous deamination, but the product is uridine, which is subject to correction by uridine-specific DNA repair pathways (49): 5-meC has long been known as a hot spot for spontaneous mutation, even at mesophilic temperatures (37,49,208). An initial screening of thermophilic bacteria showed that about half of the strains tested contained NMmeC in their DNA (54). However, by using improved methods the same authors could also demonstrate the presence of N4meC in the DNA of many mesophilic species (56), as has also been noted by others (32).
The recognition sequences for common 5-meC methyltransferases in the genomes of prokaryotes and eukaryotes and some of their viruses (for example, CCWGG in E. coli or CpG in mammalian cells) are often statistically underrepresented (55,104,175). This is generally explained by the fact that 5-meC is a hot spot for mutation, leading, when the mutations are phenotypically neutral, to loss of the methylatable sequences during evolution. However, it is also possible that some methylatable sequences were counterselected to avoid the consequences of their potential methylation on regulatory processes (104); in some phages the recognition sites for adenine methylases are also underrepresented, even though N6meA is not thought to be mutagenic (127,175). The magnitude of the mutation burden due to the deamination of 5-meC will depend on the efficiency with which the T. G mispair in DNA is detected and repaired by using the G-containing strand as the template (Fig. 2). Repair systems with precisely this specificity have been found in both prokaryotic (77, 117, 118, 182a, 220) and eukaryotic (28,210) cells. In E. coli the repair pathway is known as very short patch repair and is due to the product of the vsp gene, which is located just downstream of the dcm methylase gene (182a). Recent results from the laboratory of G. Wilson (quoted in reference 143a) indicate that ORFs with homology to vsp are found in the vicinity of the genes encoding the AluI, BsuRI, and HpaII 5-meC transferases, suggesting that very short patch-type repair may be widespread.

Type II Restriction Endonucleases That Require Two
Recognition Sites for Cleavage EcoRII was the first type II restriction endonuclease that was shown to require at least two recognition sites for endonucleolytic cleavage (99). The enzyme must bind to an activator site (which does not necessarily have to be cleaved or even be cleavable) before it will cut a cleavage site. The maximum distance between two sites in a DNA molecule which allows efficient cleavage by EcoRII is about 1 kb (146). Figure 3A shows the cleavage susceptibility of EcoRII sites located on DNA molecules of different lengths and separated by different distances. Figure 3B shows an interpretation of these results in terms of a kinetic model. The enzyme first binds reversibly to a single site on a DNA molecule. Cleavage can occur only if a second specific DNA-binding site on the enzyme is occupied before the enzyme dissociates from the first DNA molecule. The probability of having both specific binding sites simultaneously occupied will depend both on the concentration of EcoRII sites and on the size of the DNA molecules carrying those sites: rapidly diffusing oligonucleotides will be more effective at eliciting cleavage than will slowly diffusing longer DNA molecules. Very long DNA molecules diffuse so slowly that the only possibility for cleavage is for the enzyme to simultaneously bind to two sites on the same DNA molecule. The kinetics of this reaction will depend on the distance between the sites: sites that are too far apart are effectively unlinked, whereas sites that are too close together should not allow cleavage, because the DNA between them cannot form a loop. It was recently shown that NaeI can also bind simultaneously to several sites on a DNA molecule to form loops visible by electron microscopy (200).
Several authors have pointed out that the requirement of EcoRII and NaeI for simultaneous binding to two recognition sites before they will act is similar to that of enzymes responsible for site-specific recombination and for transposition (17,102,200). Future investigations will show whether these restriction endonucleases execute other biological functions besides restriction of foreign DNA. For instance, a requirement for two unmodified sites for cleavage makes it less likely that the enzyme will suicidally restrict the rare unmodified sites in chromosomal DNA that may arise by DNA repair or by incomplete methylation.
Most probably, the group of type II enzymes requiring two recognition sites for cleavage is not limited to EcoRII and NaeI. For EcoRII and NaeI the cleavage of resistant recognition sites is stimulated by adding oligonucleotides containing recognition sites or a second DNA species with a high density of recognition sites (36,99,145). Similar stimulation has also been found for the restriction enzymes AtuBI, BspMI, Cfr9I, Eco57I, HpaII, Ksp632I, NarI, SacII, and SauBMKI (144, 167, 167a), which may, therefore, also require two sites for cleavage. However, not all enzymes that cleave certain sites inefficiently have the same mechanism. The enzymes EaeI and EclXI, for instance, which also cleave certain substrates poorly, are actually inhibited by addition of site-containing oligonucleotides (166).
The fact that some restriction sites in natural DNA molecules are inefficiently cleaved can have certain practical consequences. For example, the methylation status of DNA is often assessed by digestion with pairs of enzymes that recognize the same DNA sequence but have different sensitivities to methylation: one member of the pair will cleave the methylated sequence, whereas the other will not (141). In these experiments, if the methylation-sensitive enzyme has VOL. 57,1993 resistant sites, these may be interpreted as methylated when in fact they are not. In these cases it may be helpful to use oligonucleotide stimulation of cleavage to ensure complete digestion of nonmethylated sites (167). In some experiments, in which ligation follows cleavage, for example, the presence of the cleaved stimulatory oligonucleotides may be a hindrance. For EcoRII (146) and NaeI (36a) this problem can be circumvented by the use of chemically modified oligonucleotides which stimulate cleavage of DNA but are themselves resistant to digestion.
What Is the Function of Type IIS Enzymes? Some class II R-M systems recognize asymmetrical 4to 7-bp DNA sequences and cut the DNA at a fixed distance (1 to 20 bp) outside this recognition sequence. These enzymes have been called type IIS to distinguish them from the majority of type II enzymes, which recognize symmetrical DNA sequences (for a review, see reference 194). All of the type IIS enzymes known were discovered by screening cell extracts for endonuclease activity; it is therefore not clear whether their biological function is the same as that of the more classical enzymes. The separation of the domains for DNA sequence recognition and DNA cleavage makes IIS enzymes interesting objects for studies of DNA-protein recognition (22). The genes coding several of these systems have been cloned and sequenced (213).
For classical type II R-M systems, the methylatable nucleotides are on both strands of the DNA, symmetrically disposed about the central axis of symmetry of the recognition site. The question of how the nonsymmetrical recognition sequences of type IIS systems are modified following DNA replication, which is necessary to avoid suicidal restriction of the cellular DNA, is of great interest. For most of the type IIS systems, this point has not been yet been investigated. The best-understood example is FokI, in which the methyltransferase contains two independent N6meA transferase domains, each specific for one of the two strands of the DNA (93,109,123,187). Modification in the HgaI system is accomplished by two independent 5-meC methylases, each specific for one strand of the DNA (188). It has even been reported that some type IIS systems may methylate cytosine in one strand and adenosine in the other (J. Bitinaite, quoted in reference 194). However, for MboII it has been shown that only one strand becomes methylated (22,128), and at least one other type IIS enzyme (RleAI) has neither A nor C residues in one strand of its recognition sequence and can therefore be methylated only in one strand (204). One of several ways in which this last class of systems might survive without lethal restriction of host cell DNA would be by a mechanism similar to that of the type III enzymes, which also modify only one strand of their recognition sequence (see below).

TYPE III R-M SYSTEMS
Type III R-M systems constitute the smallest group of restriction systems, with only four known members, EcoPl, EcoP15, HinfIII, and StyLTI. EcoPl and EcoP15 are coded by the P1 prophage and the related pl5B plasmid of E. coli, respectively. HinfIII is produced by Haemophilus influenzae Rf (with an isoschizomer in strain Re [147]) and is the only type III system so far found outside the family Enterobacteriaceae (88). StyLTI is coded by the chromosome of most Salmonella strains (29) and has only recently been shown to be a type III system (42)(43)(44).
The following properties are shared by all type III R-M systems. (i) The restriction enzymes contain two subunits, one of which, the mod gene product, can function alone as a modification methylase. The second subunit, the res gene product, has no enzymatic activity when not complexed with Mod (not shown for HinfIII). (ii) Restriction requires ATP, which is not hydrolyzed, and the reaction is stimulated by S-adenosylmethionine (AdoMet) (not shown for StyLTI). (iii) the recognition sequences are asymmetrical and, when modified, have a single NmeA in one strand of the DNA only, whereas the other strand has no modifications (8,42,70,130,148).

Genetics of Type III Systems
The only type III systems to be well characterized genetically are EcoPl and EcoP15. These two systems complement each other: mutational defects in one system can be complemented by wild-type alleles from the other (6). Transposon mutagenesis, studies with the purified enzymes, and DNA heteroduplex and sequence analysis showed that, contrary to some earlier reports, there are only two structural genes in these systems, with a structure reminiscent of that found within a family of type I R-M systems; that is, the res gene is highly conserved over its whole length whereas the mod gene is a mosaic of conserved and nonconserved regions (69,81). The res gene is necessary only for restriction, whereas the mod gene product provides DNA recognition for both restriction and modification as well as catalyzing modification. It is therefore a functional analog of the hsdS and hsdM gene products of type I R-M systems.
The mod genes can be divided into three roughly equal parts, the aminoand carboxy-terminal thirds of the gene, which are highly conserved between EcoPl and EcoP15, and the central third, which is completely different in the two genes (80). Several lines of evidence support the idea that the central region of the gene codes the domain of the protein that recognizes DNA sequences. First, there does not seem to be enough amino acid sequence variation in the conserved regions to allow them to recognize different DNA sequences. Second, replacing a conserved region from one gene with the equivalent region from the other did not change the specificity (52). The conserved regions of the Mod proteins are most probably involved in protein-protein interactions with the Res subunit. A similar function has also been suggested for the conserved regions of the type I hsdS gene products (58,64).
The structures of some unusual EcoPl mod mutants have been elucidated recently. These are the clear-plaque c2 mutants of phage P1 (176,177). The phenotype of these mutant phages is Mod-Res'. They cannot establish themselves as lysogens, because restriction in the absence of modification destroys the host cell chromosome. Since all infected cells are killed, regardless of whether they give rise to a productive infection, the plaques obtained are clear (171). The reason that the lytic cycle can succeed is that restriction activity is normally expressed only late after infection, when a lytically infected cell has already produced mature phage particles (7). Two independent c2 alleles were sequenced, and both mutations were found to be in the mod gene and changed two different threonine codons within the variable region to isoleucine codons (80). Both c2 mutant proteins were purified. As expected from the in vivo phenotype, they had no methylase activity and their primary defect was that they failed to bind the methyl donor, AdoMet (162). gene have also been constructed, and they encode mutants with phenotypes similar to those of the c2 mutants (162). One of these mutants also had the interesting property that, unlike the wild-type enzyme, it was no longer subject to inhibition by high concentrations of AdoMet (163). All' of these results are consistent with the idea that the variable region of the mod gene contains a DNA-binding domain and is also at least partially responsible for binding AdoMet (162).

DNA Recognition by Type HI Enzymes: Different Sequence
Requirements for Restriction and Modification All of the type III enzymes recognize asymmetrical DNA sequences, and modified DNA bears methyl groups in one strand of the DNA only (8,42,70,130,148). For example, EcoP15 recognizes CAGCAG, the second adenosyl is methylated in modified DNA (130), and no methyl groups are found in the complementary strand (70). It was early recognized that the asymmetrical methylation of modified DNA posed a conceptual problem with no immediately obvious solution (8): when a modified recognition site is replicated, the single methyl group is inherited by one daughter DNA molecule, which remains completely modified, whereas the same site in the other daughter molecule is completely unmodified and ought to be subject to restriction, which would be lethal for the cell (most other R-M systems methylate residues in both strands of the DNA, and each daughter molecule inherits one of the parental methyl groups; hemimethylated recognition sites of this type are resistant to restriction).
A solution to this problem followed the fortuitous observation that phage T3 DNA is restricted by EcoP15 whereas the DNA of its close relative, T7, is not. This was not due to a lack of EcoP15 sites in the T7 genome: phage T7 DNA has 36 sites for EcoP15 (50), which, strikingly, all have the same orientation along the DNA; that is, all of the CAGCAG sequences are in one strand of the DNA and all of the CTGCTG complementary sequences are in the other strand (175). The T3 genome has not been entirely sequenced, but the sequenced portions contain both orientations of the EcoP15 site (101). These observations led to the hypothesis that restriction requires two recognition sites that have to be in inverse orientation; sites in direct orientation would not be subject to restriction but could still be modified (Fig. 4A). This prediction was tested by using phage M13 constructs with different constellations of EcoP15 sites in both direct and inverse orientations. It was shown that any combination of sites (or a single site) could be modified, whereas only sites in inverse orientation were restricted (129). In other words, the EcoP15 endonuclease recognition site is symmetrical and is interrupted at the center of symmetry by a nonspecific spacer of variable length. Figure 4B shows the biological consequences of this model: all of the unmodified sites in freshly replicated DNA molecules are necessarily in direct orientation and thus are not subject to restriction. The model explains many early observations concerning type III restriction that at the time of publication were unexplainable. For example, densitylabeled P1-modified phage A DNA was grown for one cycle on a non-Pi-modifying host and phages of intermediate density containing DNA with one P1-modified parental DNA strand and one unmodified newly synthesized DNA strand were isolated. These phages were totally resistant to EcoPl restriction (4). A similar in vivo experiment with heteroduplex X DNA containing one EcoPl modified and one unmodified strand gave the same result (73).
(A) VOL. 57, 1993 StyLTI System The chromosomally encoded StyLTI system was difficult to characterize, largely because it proved impossible to transfer the structural genes from one strain to another by conjugation or by cloning chromosomal DNA. Conjugation resulted in extensive DNA breakdown and cell death in the recipients as a result of uncontrolled expression of restriction before the chromosome of the recipients could be modified (43). The structural genes were finally cloned in a two-step procedure, involving first cloning of the modification functions and then cloning of the restriction functions into these modifying hosts on a second, compatible vector (44). This is unusual behavior for an R-M system; the structural genes for many (but not all) restriction systems can be transferred between strains without notable killing, indicating that the expression of restriction is closely controlled.
Most recently, the StyLTI structural genes have been sequenced (41). The amino acid sequences of the StyLTI Res and Mod proteins show a good deal of homology with the EcoPl and EcoP15 proteins. In the Mod gene products, the homology is highest in the amino-and carboxy-terminal thirds of the protein and very weak in the central third (where several large gaps must be introduced to find any homology at all). This mosaic pattern of homologies strengthens the ideas about the functions of the various domains of the protein that were presented above: the central section most probably codes DNA recognition functions while the amino and carboxy termini would mediate protein-protein interactions. Homologies between the StyLTI and EcoPl Res proteins are generally weaker than those found in the Mod proteins (the EcoP15 res gene has not been sequenced). An exception is a region of about 50 amino acids in the middle of the proteins, which is very highly conserved between the two proteins and may well be involved in interactions with the Mod subunit.

DpnI and DpnH
The first restriction enzyme that was shown to require a methylated substrate was DpnI from Streptococcuspneumoniae, which cleaves the sequence GATC only if the A is methylated. Other strains of S. pneumoniae have the complementary specificity and produce DpnII, which recognizes and cleaves the same sequence only if it is not methylated (106,107). The DpnI and DpnII pair of enzymes (together with the methyltransferases associated with DpnII) present one of the clearest arguments in support of the idea that the main function of R-M systems is to protect cells from viral infection. Phages produced in S. pneumoniae strains that produce DpnII have methylated genomes and are efficiently restricted in strains that produce DpnI. Conversely, phages produced in the latter strains have nonmethylated genomes and are efficiently restricted in DpnII producers. However, S. pneumoniae is naturally transformable with high efficiency, and homologous transforming DNA is only weakly restricted in either type of strain independently of its methylation status (108). This is because during transformation of S. pneumoniae the donor DNA is taken up in single-stranded form, and neither DpnI nor DpnII is active against singlestranded DNA. Moreover, integration of the donor DNA into the chromosome involves the transient formation of a heteroduplex between the donor DNA and the recipient DNA; neither enzyme will cleave a hemimethylated recognition site. DpnII-expressing cells also favor transformation by expressing two modification methylases. One of these is a typical modification methylase, specific for doublestranded DNA, but the second enzyme is unusual in that it is very active on single-stranded DNA (34). Since transformation is thought to be important in the population biology of highly transformable organisms-S. pneumoniae can switch its specificity between DpnI and DpnII by transformation, for example (107)-it is intriguing that the R-M systems of S.
pneumoniae have evolved to have a maximum effect on phage infection with virtually no effect on homologous, transforming DNA. Methyl-dependent restriction has also been demonstrated in other bacterial species, for instance, in Streptomyces species (126).
Rediscovery of Methyl-Directed Restriction in E. coli Methylation-dependent restriction enzymes have been rediscovered in E. coli in recent years. The classical restriction enzymes operate by recognizing and destroying DNA that does not carry strain-specific modification. The new enzymes recognize and cut DNA carrying the DNA modification signature of other strains and, in consequence, are not accompanied by the equivalent of a classical modification enzyme. Three such systems have now been found in E. coli: McrA, McrBC, and Mrr (76,142,161,206). The McrBC system is the most thoroughly studied of these.
Ironically, McrBC was the first restriction system to be described in E. coli, as early as 1952 (125). This is because most phage research in those days involved the T-even phages and McrBC is active against variants of T-even phages that have nonglycosylated hydroxymethylcytosine (hmC) in their DNA. It was later shown that two independent restriction systems were active against these phage variants, and they were named RgIA and RglB (for restricts glucoseless DNA [reviewed in reference 168]). Because T-even phages were the only source of hmC-containing DNA likely to be found in E. coli, the Rgl systems were considered to be a defense mechanism specific for T-even phages (100,168). The Rgl systems were rediscovered in the late 1980s in the course of cloning experiments, when it became apparent that SmeC-containing DNA from many sources, including higher plants and animals, was restricted in E. coli and that the Rgl systems were responsible for the restriction (20,142,159,161,209,215,216). It has been formally proposed that the Mcr nomenclature (for methylcytosine restricting) is more appropriate for general use but that Rgl is still useful for discussions of T-even phage biology (158; this reference also contains a brief review of the Mcr and Mrr systems). RglA corresponds to McrA, and RglB corresponds to McrBC.

McrBC
The mcrBC (rglB) locus in E. coli K-12 is adjacent to the hsd genes that code the classical type I EcoK restriction system. Interestingly, the gene coding the Mrr system (another methyl-dependent restriction enzyme [see below]) is just on the other side of the hsd genes (76), so that genes for three independent restriction systems are clustered in this small region of the genome, which has, in consequence, been dubbed the immigration control region (89,160). There are conflicting reports in the literature concerning the organization of the mcrBC locus. However, a consensus seems to have arisen that it contains two genes, mcrB and mcrC, organized as an operon and encoding at least three polypeptides. The products of both genes are necessary for most restriction reactions (47,48,96,105,151,172,173,221,222).
McrBC restricts DNA containing 5-meC, N4meC, and hmC in specific sequence contexts (21,142,161,168). The recognition sequences have not yet been fully defined, but it seems clear that they all include R-mC, where "m" is one of the three cytosine modifications and R is either purine (161). Investigations of the enzymology of McrBC have begun, and some fascinating results have emerged. The purified enzyme contains two of the polypeptides encoded by the operon and has an absolute requirement for GTP, the only known DNase with this property. ATP, a cofactor for many nucleases, is a competitive inhibitor of the reaction (193). Work with the purified enzyme has shed further light on the DNA sequence requirements of the enzyme, but much work remains to be done. The enzyme seems to require pairs of R-mC sequences separated by 40 to 80 bp of DNA, and it is necessary only that a single strand of the DNA be methylated (193).

McrA
McrA (RglA) is coded by the e14 genetic element of many strains of E. coli K-12 (160). The e14 element is most probably a defective prophage: it can excise from the chromosome as a nonreplicating circle following UV irradiation and is then eliminated from the cells by dilution during cell division (26). The merA locus has been mapped and sequenced (79,165) and appears to code for a single polypeptide. The sequence specificity of McrA appears to be more restricted than that of McrBC, in that only DNAs methylated by the HpaII and SssI methylases (both cytosine methylases) have so far been shown to be substrates for the enzyme (79,151,161).

Mrr
The Mrr system was discovered as an activity that restricted DNA methylated by the HhaII or PstI modification enzyme (76). Both of these enzymes methylate adenosyl residues to N6meA, but it was later shown that some sequences containing 5-meC were also restricted by Mrr, and this has been referred to as McrF restriction (89,206). The mrr locus maps next to the hsd locus on the other side from mcrBC (174). The locus has been sequenced and shown to contain a single ORF (206).
In summary, the existence of the methyl-dependent restriction systems reinforces the view that the purpose of restriction is to prevent successful phage infection. E. coli K-12, for example, expends considerable metabolic energy maintaining at least four independent restriction systems containing at least eight polypeptides in readiness for a situation that most cells never encounter: invasion by foreign DNA. Despite the apparent uselessness of these restriction systems for most cells, they have not been eliminated during evolution, indicating that the selective advantage of restriction in natural populations may be much higher than one would have predicted from laboratory experiments.

Recent Developments
In this section we will describe results published since our last review on phage antirestriction mechanisms (100). A survey of the most important of these mechanisms is shown in Table 3. A large number of papers dealing with the influence of unusual bases in natural or artificial substrates on the reaction mechanism of endonucleases and methyltransferases have appeared. Studies concerning the influence of the naturally modified bases N6meA, 5-meC, and N4meC in different positions of restriction enzyme recognition sequences have been recently reviewed (91). The significance of counterselection of restriction enzyme recognition sites in phage genomes has been shown by further studies. For instance, two broad-host-range phages with very large genomes (pseudomonas phage 4ST1 and streptomycete phage FP22) have been shown to have no sites for some of the restriction enzymes found in their hosts and very few sites for other enzymes (61,71).
In the genome of coliphage T7, the recognition sites for the chromosomally encoded E. coli methylases Dam (GATC) and Dcm (CCWGG) appear only six and two times, respectively (50). Schroeder et al. (175) calculated the expected frequencies of the tetra-and pentanucleotides by Markov chain analysis on the basis of the observed occurrences of the subsequences GAT, ATC, and AT (for the Dam sequence) and CCWG, CWGG, and CWG (for the Dcm sequence) in the T7 genome. From these calculations, GATC and CCWGG are expected to occur 114 and 56 times, respectively, in the absence of counterselection. This shows that selection was directed against these particular sequences (and not against subsets of the sequences which could be involved in different selection processes), indicating that their presence was disadvantageous for the reproduction of the virus (175). In independent studies, selection against Dam sites and restriction enzyme recognition sites in the genomes of other phages of members of the Enterobacteriaceae has been demonstrated (127,178). VOL. 57,1993 In some cases, a few recognition sites for certain restriction enzymes can be tolerated in phage genomes because they do not lead to restriction. The very few EcoRII sites in the T7 and T3 genomes are not restricted, because the enzyme requires at least two sites in relatively close proximity for restriction and the distance between the EcoRII sites in the T3 and T7 genomes is too great for the enzyme to function (99). For enzymes that recognize asymmetrical sequences, the distance between sites may not be the only criterion for avoiding restriction. As discussed above for the type III enzymes, some enzymes can be sensitive to the orientation of cleavage sites. The 36 EcoP15 sites in the phage T7 genome all have the same orientation, making the DNA refractory to EcoP15 cleavage (129,175). On the other hand, restriction sites may be overrepresented in phage genomes. An example is the sequence AGACC, recognized by EcoPl, which occurs 126 times in the phage T7 genome (distributed on both DNA strands). The EcoPl recognition sequence fortuitously overlaps the site recognized by the T7 DNA primase, an enzyme essential for T7 DNA replication. This overlap is most probably the reason for the high frequency of EcoPl sites in the T7 genome and explains why T7 DNA replication is severely depressed in Pl-lysogenic cells (175).
An efficient mechanism for antirestriction has been developed by phages T3 and T7, whose 0.3 gene codes a specific inhibition protein, Ocr (100). The Ocr protein binds to type I restriction enzymes and blocks both the endonuclease and methylase activities (10,23,103,184). It is inactive against type II enzymes, both endonucleases and methylases (10). In early studies, an inhibition of the type III EcoPl restriction was demonstrated in vivo but could not be shown in vitro with the purified restriction enzyme (100). More recent studies involving a different experimental approach failed to confirm the in vivo EcoPl inhibition (134). The T3 (but not T7) 0.3 gene product has, in addition to its Ocr activity, a second enzymatic activity: the protein is an AdoMet hydrolase (186). As a result, the T3 protein not only blocks type I restriction enzymes but also has markedly inhibitory effects on type III restriction enzymes, which are stimulated by AdoMet (98).
Several phages of B. subtilis, such as SPI, SPR, 43T and pll, protect themselves from host restriction systems by phage-coded DNA methyltransferases (100). These enzymes are often multispecific; that is, they recognize several unrelated DNA sequences. The modular (and sometimes cryptic) structure of these enzymes has been worked out over the last few years by the group of Trautner and Noyer-Weidner (13,110,111,202,203,211). These results are discussed in more detail in the section on type II cytosine methyltransferases.
Recently, a DNA methyltransferase gene has been demonstrated on the genome of the Lactococcus lactis phage 4)50 (78). Unlike the Bacillus phage methyltransferase genes, which are unrelated to host cell genes, the +50 gene has clearly been picked up recently from a plasmid of its host: some 1.3 kb of DNA sequence with homologies to other methyltransferase genes is identical between the phage and the plasmid (78).
Phage A has an antirestriction mechanism called Ral that is specific for type IA R-M systems. Ral reduces restriction, but its primary effect is to stimulate the modification of completely unmodified DNA (100), a reaction that for type IA R-M systems is normally very inefficient. The ral gene has now been cloned and the antirestriction mechanism has been examined in more detail (121,223). These studies confirm that the main action of Ral is on modification and that modification by both the three-subunit restriction enzyme and the two-subunit modification enzyme are stimulated; the results are consistent with the formation of a Ral-enzyme complex with altered methylation properties. In fact, the properties of the putative complex are very similar to those of the hsdM mutants described above, which convert EcoK from a maintenance to a de novo methylase (90), and it is tempting to speculate that Ral interacts with HsdM. Previous studies had indicated that Ral might interact with the Rho transcription termination factor, and it was suggested that Rho might participate directly in restriction (45), but the present results fail to confirm this notion (121).
Finally, it should be noted that some E. coli conjugative plasmids in incompatibility groups I and N have also been shown to code antirestriction functions. These functions are active only against type I R-M systems and are coded by genes (called ard) located close to the origin of conjugative transfer, where they are expressed in the recipient bacteria very early in the conjugation process (14, 14a, 46, 53, 165a).
"Nature Red in Tooth and Claw": The Case of Phage T4 The coevolution of bacterial defenses and phage attack strategies is perhaps most clearly seen with E. coli and the T-even family of virulent phages, which includes T2, T4, and T6. A plausible case can be made for the evolutionary scenario outlined in Fig. 5. Originally, the T-even phages would have had a genome composed of normal DNA and would have been subject to restriction by the classical R-M systems of their hosts. The evolution of a hmC-containing phage genome resistant to classical restriction can be seen as a response to the presence of these classical R-M systems. The evolutionary pressure that led to the development of the Mcr (Rgl) restriction systems specific for DNA with modified cytosines was surely challenge by phages such as the T-even phages with modified cytosines in their genomes. The most recent stage in this scenario is the acquisition by the phages of the ability to glycosylate their DNA, rendering them resistant to all known restriction systems of E. coli.
The struggle between host and phage is not confined to DNA restriction. Certain strains of E. coli have a restriction system specific for T-even phages that operates on the RNA level. This enzyme, theprrC gene product, is present but has no activity in uninfected cells. The activity is unmasked on infection with a T-even phage and proceeds to make a specific cleavage in the anticodon of the host-encoded tRNALYS. This inactivates protein synthesis and thus prevents phage multiplication. Phage T4 encodes two enzymes, polynucleotide kinase and RNA ligase, whose only known function in the T4 life cycle is to repair the damage caused by the anticodon nuclease (83,116). A surprising finding was that theprrC gene is inserted into an hsd operon encoding an active type I R-M system (Fig. 1) (119). Moreover, the PrrC protein can be immunoprecipitated with anti-Hsd serum, indicating that it is physically complexed with the restriction enzyme. Both DNA-and RNA-based restriction systems are thus tightly integrated at both the genetic and the protein levels (1). phages severely, whereas beneficial transformation is hardly affected. Other roles for restriction in the cellular economy are more difficult to document. For example, restriction may facilitate recombination by generating recombinogenic double-stranded breaks in homologous incoming DNA (181), and it has been suggested that type I R-M systems would be particularly effective in this role (153).

CONCLUDING REMARKS
It should not be forgotten that a small fraction of invading DNA molecules evade restriction and become modified. Thus, although restriction provides a barrier to genetic mobility, the barrier is leaky, allowing the possibility of a low rate of evolution by horizontal gene transfer. ACKNOWLEDGMENTS We thank the many members of the nuclease community who sent us manuscripts prior to publication and Mario Noyer-Weidner for a critical appraisal of the manuscript of this review. Special thanks are due to Rich Roberts, who maintains a comprehensive data base of the restriction literature, which he makes freely available to the scientific community. This review would have been much more difficult to prepare without access to this data base.
Work from Basel has been supported by grants from the Swiss National Science Foundation. Recent work from Berlin was supported by the Deutsche Forschungsgemeinschaft (Kr 1293/1-1) and the Federal Minister for Research and Technology (grant 0319854 A).