Purification and DNA-binding properties of FIS and Cin, two proteins required for the bacteriophage P1 site-specific recombination system, cin.

An Escherichia coli chromosomally coded factor termed FIS (Factor for Inversion Stimulation) stimulates the Cin protein-mediated, site-specific DNA inversion system of bacteriophage P1 more than 500-fold. We have purified FIS and the recombinase Cin, and studied the inversion reaction in vitro. DNA footprinting studies with DNase I showed that Cin specifically binds to the recombination site, called cix. FIS does not bind to cix sites but does bind to a recombinational enhancer sequence that is required in cis for efficient recombination. FIS also binds specifically to sequences outside the enhancer, as well as to sequences unrelated to Cin inversion. On the basis of these data, we discuss the possibility of additional functions for FIS in E. coli.

Abortive infection of Escherichia coli F+ cells by bacteriophage T7 requires ribosomal misreading.

The use of different precisely mapped T3/T7 recombinants strengthens the conclusion that abortive infection by T7 of F plasmid-carrying cells is due to the nucleotide sequence at the end of the T7 gene 1. Furthermore, we demonstrate that the exclusion requires suppression of ochre stop codons, a phenomenon that occurs with low frequency in wild-type cells due to ribosomal misreading. The introduction of rspL mutations in which ribosomal misreading is reduced alleviates the exclusion and the presence of ochre tRNA suppressors increases its severity.

Regulation of the activity of the type IC EcoR124I restriction enzyme.

Restriction-modification (R-M) systems must regulate the expression of their genes so that the chromosomal genome is modified at all times by the methyltransferase to protect the host cell from the potential lethal action of the cognate restriction endonuclease. Since type I R-M systems can be transferred to non-modified Escherichia coli cells by conjugation or transformation without killing the recipient, they must have some means to regulate their restriction activity upon entering a new host cell to avoid restriction of unprotected host DNA and cell death. This is especially true for EcoR124I, a type IC family member, which is coded for by a conjugative plasmid. Control of EcoR124I restriction activity is most likely at the post-translational level as the transfer of the EcoR124I system into a recipient cell that already expressed the HsdR subunit of this system was not a lethal event. Additionally, the kinetics of restriction activity upon transfer of the genes coding for the EcoR124I RM system to a recipient cell are the same, irrespective of the modification state of the recipient cell or the presence or absence of the EcoR124I HsdR subunit in the new host cells. The mechanism controlling the restriction activity of a type IC R-M system upon transfer to a new host cell is different from that controlling the chromosomally coded type IA and IB R-M systems. The previously discovered hsdC mutant, which affects the establishment of the type IA system EcoKI, was shown to affect the establishment of the type IB system EcoAI, but to have no influence on EcoR124I.

DNA supercoiling during ATP-dependent DNA translocation by the type I restriction enzyme EcoAI.

Type I restriction enzymes cleave DNA at non-specific sites far from their recognition sequence as a consequence of ATP-dependent DNA translocation past the enzyme. During this reaction, the enzyme remains bound to the recognition sequence and translocates DNA towards itself simultaneously from both directions, generating DNA loops, which appear to be supercoiled when visualised by electron microscopy. To further investigate the mechanism of DNA translocation by type I restriction enzymes, we have probed the reaction intermediates with DNA topoisomerases. A DNA cleavage-deficient mutant of EcoAI, which has normal DNA translocation and ATPase activities, was used in these DNA supercoiling assays. In the presence of eubacterial DNA topoisomerase I, which specifically removes negative supercoils, the EcoAI mutant introduced positive supercoils into relaxed plasmid DNA substrate in a reaction dependent on ATP hydrolysis. The same DNA supercoiling activity followed by DNA cleavage was observed with the wild-type EcoAI endonuclease. Positive supercoils were not seen when eubacterial DNA topoisomerase I was replaced by eukaryotic DNA topoisomerase I, which removes both positive and negative supercoils. Furthermore, addition of eukaryotic DNA topoisomerase I to the product of the supercoiling reaction resulted in its rapid relaxation. These results are consistent with a model in which EcoAI translocation along the helical path of closed circular DNA duplex simultaneously generates positive supercoils ahead and negative supercoils behind the moving complex in the contracting and expanding DNA loops, respectively. In addition, we show that the highly positively supercoiled DNA generated by the EcoAI mutant is cleaved by EcoAI wild-type endonuclease much more slowly than relaxed DNA. This suggests that the topological changes in the DNA substrate associated with DNA translocation by type I restriction enzymes do not appear to be the trigger for DNA cleavage.

The DNA recognition subunit of the type IB restriction-modification enzyme EcoAI tolerates circular permutions of its polypeptide chain.

The DNA specificity subunit (HsdS) of type I restriction-modification enzymes is composed of two independent target recognition domains and several regions whose amino acid sequence is conserved within an enzyme family. The conserved regions participate in intersubunit interactions with two modification subunits (HsdM) and two restriction subunits (HsdR) to form the complete endonuclease. It has been proposed that the domains of the HsdS subunit have a circular organisation providing the required symmetry for their interaction with the other subunits and with the bipartite DNA target. To test this model, we circularly permuted the HsdS subunit of the type IB R-M enzyme EcoAI at the DNA level by direct linkage of codons for original termini and introduction of new termini elsewhere along the N-terminal and central conserved regions. By analysing the activity of mutant enzymes, two circularly permuted variants of HsdS that had termini located at equivalent positions in the N-terminal and central repeats, respectively, were found to fold into a functional DNA recognition subunit with wild-type specificity, suggesting a close proximity of the N and C termini in the native protein. The wild-type HsdS subunit was purified to homogeneity and shown to form a stable trimeric complex with HsdM, M2S1, which was fully active as a DNA methyltransferase. Gel electrophoretic mobility shift assays revealed that the HsdS protein alone was not able to form a specific complex with a 30-mer oligoduplex containing a single EcoAI recognition site. However, addition of stoichiometric amounts of HsdM to HsdS led to efficient specific DNA binding. Our data provide evidence for the circular organisation of domains of the HsdS subunit. In addition, they suggest a possible role of HsdM subunits in the formation of this structure.

ATPase activity of the type IC restriction-modification system EcoR124II.

We have investigated the ATPase activity of the type IC restriction-modification (R-M) system EcoR124II. As with all type I R-M systems EcoR 124II requires ATP hydrolysis to cut DNA. We determined the KM for ATP to be 10(-5) to 10(-4) M. By measuring ATP hydrolysis under different conditions and by simultaneously monitoring DNA restriction, methylation and ATP hydrolysis we propose that the order of events during restriction is: (1) binding of EcoR124II to a non-methylated recognition sequence, (2) start of DNA-dependent ATP hydrolysis which continues even after restriction is complete, (3) restriction of DNA, (4) methylation of the product. Non-cleavable DNA substrates, such as recognition site containing oligonucleotides, also support ATP hydrolysis. Methylation can also occur prior to ATP hydrolysis and prevent DNA degradation.

Posttranscriptional regulation of EcoP1I and EcoP15I restriction activity.

Efficient establishment of a DNA restriction-modification (R-M) system in a non-modified cell requires a tight control of the potentially lethal activity of the restriction enzyme. The type III R-M systems EcoP1I and EcoP15I can be transferred to non-modified Escherichia coli cells by transfection, conjugation or transformation and become established without difficulty. Modification activity is expressed immediately after the R-M genes enter the cell, whereas the expression of restriction activity is delayed until complete protection of the cellular DNA is achieved by methylation. We have shown by Western blot analysis that the expression of the modification polypeptide subunit positively regulates the amount of restriction subunit present in the cell. The finding that ribosomal alterations affected the expression of restriction activity pointed to additional control at the translational level. The analysis of EcoP1I expression in E. coli strains mutated in either of the ribosomal proteins S12 (rpsL) or S4 (rpsD) suggests that the level of in vivo restriction activity can be modulated both by a decrease in the efficiency of translation and by varying ribosomal accuracy conditions. In addition, we have preliminary evidence from in vivo gene fusion studies that the res gene may code for more than one gene product.

EcoA: the first member of a new family of type I restriction modification systems. Gene organization and enzymatic activities.

The characterization of the EcoA restriction-modification enzymes from Escherichia coli 15T- is described. The reactions catalysed by these enzymes are very similar to those catalysed by the classical type I restriction and modification enzymes, a family of genetically related proteins. The detailed mechanisms, particularly for DNA modification, differ. The genetic and transcriptional organizations are also very similar to those of the classical systems, despite the fact that EcoA is not allelic to the others. We demonstrate that the expression of the EcoA genes is controlled following conjugative transfer to other strains in such a way that no lethality is observed, probably because the recipient chromosome is completely modified before restriction activity is expressed.

DNA cleavage by the type IC restriction-modification enzyme EcoR124II.

Type I restriction-modification systems bind to non-palindromic, bipartite recognition sequences. Although these enzymes methylate specific adenine residues within their recognition sequences, they cut DNA at sites up to several thousand base-pairs away. We have investigated the mechanism of how EcoR124II, a type IC restriction-modification system, selects the cleavage site. Restriction studies with different DNA constructs revealed that circular DNA requires only one non-methylated recognition sequence to be cut, whereas linear DNA needs at least two such sites. Cleavage of linear DNA is independent of site orientation. Further investigations of the linear substrates revealed a mechanism whereby the double-strand break is introduced between two recognition sequences. We propose a model for the selection of restriction sites by type I enzymes where two EcoR124II complexes bind to two recognition sequences. Lack of methylation at a site stimulates the enzyme to translocate DNA on both sides of the recognition sequence. Thus the two complexes approach each other and, at the point where they meet, they interact to introduce a double-strand break in the DNA.

The McrBC endonuclease translocates DNA in a reaction dependent on GTP hydrolysis.

McrBC specifically recognizes and cleaves methylated DNA in a reaction dependent on GTP hydrolysis. DNA cleavage requires at least two recognition sites that are optimally separated by 40-80 bp, but can be spaced as far as 3 kb apart. The nature of the communication between two recognition sites was analyzed on DNA substrates containing one or two recognition sites. DNA cleavage of circular DNA required only one methylated recognition site, whereas the linearized form of this substrate was not cleaved. However, the linearized substrate was cleaved if a Lac repressor was bound adjacent to the recognition site. These results suggest a model in which communication between two remote sites is accomplished by DNA translocation rather than looping. A mutant protein with defective GTPase activity cleaved substrates with closely spaced recognition sites, but not substrates where the sites were further apart. This indicates that McrBC translocates DNA in a reaction dependent on GTP hydrolysis. We suggest that DNA cleavage occurs by the encounter of two DNA-translocating McrBC complexes, or can be triggered by non-specific physical obstacles like the Lac repressor bound on the enzyme's path along DNA. Our results indicate that McrBC belongs to the general class of DNA "motor proteins", which use the free energy associated with nucleoside 5'-triphosphate hydrolysis to translocate along DNA.

The Escherichia coli prr region encodes a functional type IC DNA restriction system closely integrated with an anticodon nuclease gene.

The prr locus was originally described as coding a ribonuclease that is activated after phage T4 infection to cut within the anticodon of a specific tRNA, inactivating protein synthesis and thus blocking phage development. Wild-type T4 phage has two genes coding the enzymes polynucleotide kinase and RNA ligase, whose only function seems to be to repair the damage done by the anticodon nuclease. As the only apparent function of the prr ribonuclease is to combat phage infection, it can be considered as an RNA-based restriction enzyme. In non-infected cells, the prr enzyme is kept inactive in a complex with three other proteins which were predicted on the basis of DNA homologies to be the subunits of a type IC DNA restriction and modification system. Unlike other type IC systems so far characterized, prr is chromosomally rather than plasmid coded. However, sequences upstream from prr also have homology with sequences from the plasmid R124 and the prophage P1. We have now investigated the prr system and shown that it is indeed a bona fide type IC system which we call EcoprrI, and which is active both in vivo and in vitro. The system is fully functional even in the absence of the anticodon nuclease and seems to be a typical type I enzyme. EcoprrI recognizes the sequence CCA(N7)RTGC. One peculiarity is that, with low efficiency, EcoprrI will recognize and methylate variants of its recognition sequence such as CCT(N7)ATGC, which is methylated in one strand of the DNA only.

Subunit assembly and mode of DNA cleavage of the type III restriction endonucleases EcoP1I and EcoP15I.

DNA cleavage by type III restriction endonucleases requires two inversely oriented asymmetric recognition sequences and results from ATP-dependent DNA translocation and collision of two enzyme molecules. Here, we characterized the structure and mode of action of the related EcoP1I and EcoP15I enzymes. Analytical ultracentrifugation and gel quantification revealed a common Res(2)Mod(2) subunit stoichiometry. Single alanine substitutions in the putative nuclease active site of ResP1 and ResP15 abolished DNA but not ATP hydrolysis, whilst a substitution in helicase motif VI abolished both activities. Positively supercoiled DNA substrates containing a pair of inversely oriented recognition sites were cleaved inefficiently, whereas the corresponding relaxed and negatively supercoiled substrates were cleaved efficiently, suggesting that DNA overtwisting impedes the convergence of the translocating enzymes. EcoP1I and EcoP15I could co-operate in DNA cleavage on circular substrate containing several EcoP1I sites inversely oriented to a single EcoP15I site; cleavage occurred predominantly at the EcoP15I site. EcoP15I alone showed nicking activity on these molecules, cutting exclusively the top DNA strand at its recognition site. This activity was dependent on enzyme concentration and local DNA sequence. The EcoP1I nuclease mutant greatly stimulated the EcoP15I nicking activity, while the EcoP1I motif VI mutant did not. Moreover, combining an EcoP15I nuclease mutant with wild-type EcoP1I resulted in cutting the bottom DNA strand at the EcoP15I site. These data suggest that double-strand breaks result from top strand cleavage by a Res subunit proximal to the site of cleavage, whilst bottom strand cleavage is catalysed by a Res subunit supplied in trans by the distal endonuclease in the collision complex.

Cloning, over-expression and the catalytic properties of the EcoP15 modification methylase from Escherichia coli.

The EcoP15 modification methylase gene from the p15B plasmid of Escherichia coli 15T-has been cloned and expressed at high levels in a plasmid vector system. We have purified the enzyme to near homogeneity in large amounts and have studied some of its enzymatic properties. Initial rates of methyl transfer are first order in methylase concentration and, with pUC19 DNA as substrate, the reaction proceeds by a random mechanism in which either DNA or S-adenosylmethionine can bind to the free enzyme. After methyltransfer to DNA, the methylated DNA and S-adenosylhomocysteine appear to dissociate in random order. As expected in such a mechanism, S-adenosylhomocysteine is a non-competitive inhibitor by S-adenosylmethionine at concentrations not much above its KM suggests that release of methylated DNA may be the rate-limiting step. This suggestion is strengthened by the fact that a mutant of the closely related EcoP1 does not show such substrate inhibition.

Methyl-specific DNA binding by McrBC, a modification-dependent restriction enzyme.

McrBC, a GTP-requiring, modification-dependent endonuclease of Escherichia coli K-12, specifically recognizes DNA sites of the form 5' R(m)C 3'. DNA cleavage normally requires translocation-mediated coordination between two such recognition elements at distinct sites. We have investigated assembly of the cleavage-competent complex with gel-shift and DNase I footprint analysis. In the gel-shift system, McrB(L) binding resulted in a fast-migrating specific shifted band, in a manner requiring both GTP and Mg(2+). The binding was specific for methylated DNA and responded to local sequence changes in the same way that cleavage does. Single-stranded DNA competed for McrB(L)-binding in a modification and sequence-specific fashion. A supershifted species was formed in the presence of McrC and GTPgammaS. DNase I footprint analysis showed modest cooperativity in binding to two sites, and a two-site substrate displayed protection in non-specific spacer DNA in addition to the recognition elements. The addition of McrC did not affect the footprint obtained. We propose that McrC effects a conformational change in the complex rather than a reorganization of the DNA:protein interface.

Two type I restriction enzymes from Salmonella species. Purification and DNA recognition sequences.

We have purified the type I restriction enzymes SB and SP from Salmonella typhimurium and S. potsdam, respectively, and determined the DNA sequences that they recognize. These sequences resemble those previously determined for the type I enzymes, EcoB, EcoK and EcoA, in that the specific part of the sequence is divided into two domains by a spacer of non-specific sequence that has a fixed length for each enzyme. Two main differences from the previously determined sequences are seen. Both of the new sequences are degenerate and one of them, SB, has one trinucleotide and one pentanucleotide-specific domain rather than the trinucleotide and tetranucleotide domains seen for all of the other enzymes. The only conserved features of the recognition sequences are the adenosyl residues that are methylated in the modification reaction. For all of the enzymes these are situated ten or 11 base-pairs apart, one on each strand of the DNA. This suggests that the enzymes bind to DNA along one face of the double helix making protein-DNA interaction in two successive major grooves with most of the non-specific spacer sequence in the intervening minor groove.

Gene 68, a new bacteriophage T4 gene which codes for the 17K prohead core protein is involved in head size determination.

We have identified the gene for a major component of the prohead core of bacteriophage T4, the 17K protein. The gene, which we call gene 68, lies between genes 67 and 21 in the major cluster of T4 head genes. All of the genes in this region of the T4 genome have overlapping initiation and termination codons with the sequence T-A-A-T-G. We present the DNA sequence of the gene and show that it codes for a protein containing 141 amino acids with an acidic amino-terminal half and a basic carboxyl terminus. Antibodies prepared against the 17K protein were used to show that it is cleaved by the phage-coded gp21 protease during head maturation and that most of the protein leaves the head after cleavage. A frameshift mutation of the gene was constructed in vitro and recombined back into the phage genome. The mutated phages had a drastically reduced burst size and about half of the particles produced were morphologically abnormal, having isometric rather than prolate heads. Thus, the 17K protein is involved in head shape determination but is only semi-essential for T4 growth.

DNA restriction--modification enzymes of phage P1 and plasmid p15B. Subunit functions and structural homologies.

We have purified the type III restriction enzymes EcoP1 and EcoP15 to homogeneity from bacteria that contain the structural genes for the enzymes cloned on small, multicopy plasmids and which overproduce the enzymes. Both of the enzymes contain two different subunits. The molecular weights of the subunits are the same for both enzymes and antibodies prepared against one enzyme cross-react with both subunits of the other. Bacteria containing a plasmid derivative in which a large part of one of the structural genes has been deleted have a restriction- modification+ phenotype and contain only the smaller of the two subunits. This subunit therefore must be the one that both recognizes the specific DNA sequence and methylates it in the modification reaction (the restriction enzyme itself also acts as a modification methylase). We have purified the P1 and P15 modification subunits from these deletion derivatives and have shown that in vitro they have the expected properties: they are sequence-specific modification methylases. In addition, we have demonstrated that strains carrying the full restriction/modification system also contain a pool of free modification subunits that might be responsible for in vivo modification.

Determination of the cleavage site of the phage T4 prohead protease in gene product 68. Influence of protein secondary structure on cleavage specificity.

The cleavage site of the T4 prohead protease in gene product 68 of bacteriophage T4 has been determined by direct protein sequencing. It is located close to the carboxy-terminal end of a predicted alpha-helix in the sequence Asn-Val-Glu-Ala between the Glu and Ala residues. Secondary structure seems to be more important in determining cleavage than the presence of an aliphatic amino acid three residues before the cleavage site that was proposed earlier. In this case, that position is occupied by Asn, a hydrophilic residue. A second potentially cleavable Glu-Ala is found five residues after the cleaved sequence and this is preceded by an Ile at the -3 position. Despite this, the sequences of the amino and carboxyl termini of the uncleaved protein are identical to those previously proposed from an analysis of the DNA sequence of the gene.

Amber mutants in gene 67 of phage T4. Effects on formation and shape determination of the head.

Two amber mutations in gene 67 of bacteriophage T4 were constructed by oligonucleotide-directed mutagenesis and the resulting mutated genes were recombined back into the phage genome and their phenotype was studied. The 67amK1 mutation is close to the amino terminus of the gene, and phage carrying this mutation are unable to form plaques on suppressor-negative hosts. A second mutation, 67amK2, which lies in the middle of the gene, three codons N-terminal to a proteolytic cleavage site, produces a small number of viable phage particles. In suppressor-negative hosts, both mutants produce polyheads and proheads. 67amK1 assembles only few proheads that have a disorganized core structure, as judged from thin sections of infected cells. The proheads and the mature phages of both mutants are mainly isometric rather than having the usual prolate shape. Depending on the 67 mutant and the host, between 20% and 73% of the particles that are produced are isometric, and 1 to 10% are two-tailed biprolate particles. 67amK2 phages grown on a supD suppressor strain that inserts serine in place of the wild-type leucine do not contain gp67* derived from gene product 67 (gp67) by proteolytic cleavage. This demonstrates the importance of the correct amino acid at this position in the protein. Other abnormalities in these 67amK2 phages are the presence of uncleaved scaffolding core proteins (IPIII and gp68), indicating a structural alteration in the prohead scaffold, resulting in only partial cleavage. In wild-type phages these proteins are found in the head only in the cleaved form. With double-mutants of 67 with mutations in the major shell protein gp23 no naked scaffolding cores were found, confirming the necessity of gp67 for the assembly or persistence of a "normal" core.

KpnAI, a new type I restriction-modification system in Klebsiella pneumoniae.

The KpnAI restriction-modification (R-M) system has been identified in Klebsiella pneumoniae strain M5a1. The restriction gene of KpnAI was first cloned into pBR322 using an r-m+ M5a1 derivative and phage SBS for screening. Subsequently, an adjacent DNA fragment showing modification activity was cloned into pUC19. A total of 7.2 kb DNA sequencing data revealed three open reading frames, corresponding to hsdR, hsdM and hsdS genes of type I R-M systems. The predicted hsdR, hsdM and hsdS-coded peptides shared 95%, 98% and 44% identity, respectively, with the corresponding peptides of the recently identified StySBLI system, a prototype of the type ID family. This high homology suggests that KpnAI is also a member of the type ID family. The KpnAI system seems to be the first type I system identified in Klebsiella species.