The Werner syndrome protein is required for recruitment of chromatin assembly factor 1 following DNA damage.

The Werner syndrome protein (WRN) and chromatin assembly factor 1 (CAF-1) are both involved in the maintenance of genome stability. In response to DNA-damaging signals, both of these proteins relocate to sites where DNA synthesis occurs. However, the interaction between WRN and CAF-1 has not yet been investigated. In this report, we show that WRN interacts physically with the largest subunit of CAF-1, hp150, in vitro and in vivo. Although hp150 does not alter WRN catalytic activities in vitro, and the chromatin assembly activity of CAF-1 is not affected in the absence of WRN in vivo, this interaction may have an important role during the cellular response to DNA replication fork blockage and/or DNA damage signals. In hp150 RNA-mediated interference (RNAi) knockdown cells, WRN partially formed foci following hydroxyurea (HU) treatment. However, in the absence of WRN, hp150 did not relocate to form foci following exposure to HU and ultraviolet light. Thus, our results demonstrate that WRN responds to DNA damage before CAF-1 and suggest that WRN may recruit CAF-1, via interaction with hp150, to DNA damage sites during DNA synthesis.

Characterization of an EcoR124I restriction-modification enzyme produced from a deleted form of the DNA-binding subunit, which results in a novel DNA specificity.

We purified and characterized both the methyltransferase and the endonuclease containing the HsdS delta 50 subunit (type I restriction endonucleases are composed of three subunits--HsdR required for restriction, HsdM required for methylation and HsdS responsible for DNA recognition) produced from the deletion mutation hsdS delta 50 of the type IC R-M system EcoR 124I; this mutant subunit lacks the C-terminal 163 residues of HsdS and produces a novel DNA specificity. Analysis of the purified HsDs delta 50 subunit indicated that during purification it is subject to partial proteolysis resulting in removal of approximately 1 kDa of the polypeptide at the C-terminus. This proteolysis prevented the purification of further deletion mutants, which were determined as having a novel DNA specificity in vivo. After biochemical characterization of the mutant DNA methyltransferase (MTase) and restriction endonuclease we found only one difference comparing with the wild-type enzyme--a significantly higher binding affinity of the MTase for the two substrates of hemimethylated and fully methylated DNA. This indicates that MTase delta 50 is less able to discriminate the methylation status of the DNA during its binding. However, the mutant MTase still preferred hemimethylated DNA as the substrate for methylation. We fused the hsdM and hsdS delta 50 genes and showed that the HsdM-HsdS delta 50 fusion protein is capable of dimerization confirming the model for assembly of this deletion mutant.

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.

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.

Two temperature-sensitive mutations in the DNA binding subunit of EcoKI with differing properties.

Two temperature-sensitive mutations in the hsdS gene, which encodes the DNA specificity subunit of the type IA restriction-modification system EcoKI, designated Sts1 (Ser(340)Phe) and Sts2 (Ala(204)Thr) 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 degrees 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 degrees C. Unlike the HsdSts1 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.

Single amino acid substitutions in the HsdR subunit of the type IB restriction enzyme EcoAI uncouple the DNA translocation and DNA cleavage activities of the enzyme.

Type I restriction enzymes bind to specific DNA sequences but subsequently translocate non-specific DNA past the complex in a reaction coupled to ATP hydrolysis and cleave DNA at any barrier that can halt the translocation process. The restriction subunit of these enzymes, HsdR, contains a cluster of seven amino acid sequence motifs typical of helicase superfamily II, that are believed to be relevant to the ATP-dependent DNA translocation. Alignment of all available HsdR sequences reveals an additional conserved region at the protein N-terminus with a consensus sequence reminiscent of the P-D.(D/E)-X-K catalytic motif of many type II restriction enzymes. To investigate the role of these conserved residues, we have produced mutants of the type IB restriction enzyme Eco AI. We have found that single alanine substitutions at Asp-61, Glu-76 and Lys-78 residues of the HsdR subunit abolished the enzyme's restriction activity but had no effect on its ATPase and DNA translocation activities, suggesting that these residues are part of the active site for DNA cleavage.

DNA translocation blockage, a general mechanism of cleavage site selection by type I restriction enzymes.

Type I restriction enzymes bind to a specific DNA sequence and subsequently translocate DNA past the complex to reach a non-specific cleavage site. We have examined several potential blocks to DNA translocation, such as positive supercoiling or a Holliday junction, for their ability to trigger DNA cleavage by type I restriction enzymes. Introduction of positive supercoiling into plasmid DNA did not have a significant effect on the rate of DNA cleavage by EcoAI endonuclease nor on the enzyme's ability to select cleavage sites randomly throughout the DNA molecule. Thus, positive supercoiling does not prevent DNA translocation. EcoR124II endonuclease cleaved DNA at Holliday junctions present on both linear and negatively supercoiled substrates. The latter substrate was cleaved by a single enzyme molecule at two sites, one on either side of the junction, consistent with a bi-directional translocation model. Linear DNA molecules with two recognition sites for endonucleases from different type I families were cut between the sites when both enzymes were added simultaneously but not when a single enzyme was added. We propose that type I restriction enzymes can track along a DNA substrate irrespective of its topology and cleave DNA at any barrier that is able to halt the translocation process.

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.

Analysis of the subunit assembly of the typeIC restriction-modification enzyme EcoR124I.

Type I restriction-modification (R-M) enzymes are composed of three different subunits, of which HsdS determines DNA specificity, HsdM is responsible for DNA methylation and HsdR is required for restriction. The HsdM and HsdS subunits can also form an independent DNA methyltransferase with a subunit stoichiometry of M2S1. We found that the purified Eco R124I R-M enzyme was a mixture of two species as detected by the presence of two differently migrating specific DNA-protein complexes in a gel retardation assay. An analysis of protein subunits isolated from the complexes indicated that the larger species had a stoichiometry of R2M2S1and the smaller species had a stoichiometry of R1M2S1. In vitro analysis of subunit assembly revealed that while binding of the first HsdR subunit to the M2S1complex was very tight, the second HsdR subunit was bound weakly and it dissociated from the R1M2S1complex with an apparent K d of approximately 2.4 x 10(-7) M. Functional assays have shown that only the R2M2S1complex is capable of DNA cleavage, however, the R1M2S1complex retains ATPase activity. The relevance of this situation is discussed in terms of the regulation of restriction activity in vivo upon conjugative transfer of a plasmid-born R-M system into an unmodified host cell.

Protein-protein and protein-DNA interactions in the type I restriction endonuclease R.EcoR124I.

The type I restriction-modification system EcoR124I recognizes and binds to the split DNA recognition sequence 5'-GAAN(6)RTCG-3'. The methyltransferase, consisting of HsdM and HsdS subunits with the composition M2S, can interact with one or more subunits of the HsdR subunit to form the endonuclease. The interaction of the methyltransferase with HsdR has been investigated by surface plasmon resonance, showing that there are two non-equivalent binding sites for HsdR which differ in binding affinity by at least two orders of magnitude. DNA footprinting experiments using Exonuclease III suggest that the addition of HsdR to the methyltransferase (at a stoichiometry of either 1:1 or 2:1) increases the stability of the resulting DNA-protein complex but does not increase the size of the footprint. More extensive in situ footprinting experiments using copper-phenanthroline on the DNA-protein complexes formed by M2S, R1M2S and R2M2S also show no difference in the detailed cleavage pattern, with approximately 18 nucleotides protected on both strands in each complex. Thus the HsdR subunit(s) of the endonuclease stabilise the interaction of the M2S complex with DNA, but do not directly contribute to DNA binding. In addition, the thymidine nucleotide in the tetranucleotide recognition sequence GTCG is hyper-reactive to cleavage in each case, suggesting that the DNA structure in this region is altered in these complexes.

Selection of non-specific DNA cleavage sites by the type IC restriction endonuclease EcoR124I.

The Type IC restriction endonuclease EcoR124I binds specifically to its recognition sequence but subsequently translocates non-specific DNA past the complex in an ATP-dependent mechanism. The enzyme thus has the potential to cleave DNA at loci distant from the recognition site. We have scrutinised the link between translocation and cleavage on linear and circular DNA substrates. On linear DNA carrying two recognition sites, the majority of cleavages at loci distant from the recognition site occurred between the two sites, regardless of the inter-site distance or relative orientations. On circular DNA carrying one site, distant cleavages occurred throughout the DNA but an equivalent linear molecule underwent considerably fewer cleavages at distant loci. These results agree with published models for DNA tracking. However, on every molecule investigated, discrete cleavage sites were also observed within +/-250 bp of the recognition sites. The localised cleavages were not confined to particular DNA sequences and were independent of DNA topology. We propose a model to account for both distant and localised cleavage events. The conformation of the DNA loop extruded during tracking may result in two DNA segments being held in proximity to the restriction moiety on the protein, one close to the EcoR124I site and another distant from the site: cleavage may occur in either segment. Alternatively, the cutting of DNA close to recognition sites may be the result of multiple nicks being generated in the expanding loop before any extensive translocation.

The HsdR subunit of R.EcoR124II: cloning and over-expression of the gene and unexpected properties of the subunit.

Type I restriction endonucleases are composed of three subunits, HsdR, HsdM and HsdS. The HsdR subunit is absolutely required for restriction activity; while an independent methylase is composed of HsdM and HsdS subunits. DNA cleavage is associated with a powerful ATPase activity during which DNA is translocated by the enzyme prior to cleavage. The presence of a Walker type I ATP-binding site within the HsdR subunit suggested that the subunit may be capable of independent enzymatic activity. Therefore, we have, for the first time, cloned and over-expressed the hsdRgene of the type IC restriction endonuclease EcoR124II. The purified HsdR subunit was found to be a soluble monomeric protein capable of DNA- and Mg2+-dependent ATP hydrolysis. The subunit was found to have a weak nuclease activity both in vivo and in vitro, and to bind plasmid DNA; although was not capable of binding a DNA oligoduplex. We were also able to reconstitute the fully active endonuclease from purified M. EcoR124I and HsdR. This is the first clear demonstration that the HsdR subunit of a type I restriction endonuclease is capable of independent enzyme activity, and suggests a mechanism for the evolution of the endonuclease from the independent methylase.

Repercussions of DNA tracking by the type IC restriction endonuclease EcoR124I on linear, circular and catenated substrates.

Type I restriction endonucleases such as EcoR124I cleave DNA at undefined loci, distant from their recognition sequences, by a mechanism that involves the enzyme tracking along the DNA between recognition and cleavage sites. This mechanism was examined on plasmids that carried recognition sites for EcoR124I and recombination sites for resolvase, the latter to create DNA catenanes. Supercoiled substrates with either one or two restriction sites were linearized by EcoR124I at similar rates, although the two-site molecule underwent further cleavage more readily than the one-site DNA. The catenane from the plasmid with one EcoR124I site, carrying the site on the smaller of the two rings, was cleaved by EcoR124I exclusively in the small ring, and this underwent multiple cleavage akin to the two-site plasmid. Linear substrates derived from the plasmids were cleaved by EcoR124I at very slow rates. The communication between recognition and cleavage sites therefore cannot stem from random looping. Instead, it must follow the DNA contour between the sites. On a circular DNA, the translocation of non-specific DNA past the specifically bound protein should increase negative supercoiling in one domain and decrease it in the other. The ensuing topological barrier may be the trigger for DNA cleavage.

The type I restriction endonuclease R.EcoR124I: over-production and biochemical properties.

In this paper we describe a two-plasmid system which allows over-production of the R.EcoR124I restriction endonuclease. The endonuclease has been purified to homogeneity in milligram amounts and has been shown to be fully active for both restriction and modification. Unexpectedly, the enzyme was found to require only ATP and Mg2+ for ATPase activity and DNA cleavage; S-adenosyl methionine (SAM), which has been described as a cofactor of type I restriction enzymes, is not required by R.EcoR124I. However, SAM was found to stimulate the rate of ATPase activity and DNA cleavage. This may occur through an increase in specific DNA binding by R.EcoR124I in the presence of SAM, as indicated by our surface plasmon resonance experiments. These functional differences from the well described R.EcoKI restriction endonuclease are reflected in a possible structural difference between the two enzymes, namely that the stoichiometry of R.EcoR124I appears to be R1M2S1 while that of R.EcoKI is R2M2S1. Supercoiled DNA with one or two SR124I recognition sites is cleaved by the same mechanism inferring co-operation between specifically bound and excess enzymes. Nicked-circle DNA is an intermediate of cleavage reaction. Cleavage of DNA was inhibited by an increased degree of negative supercoiling, which may reflect an increased difficulty for the enzyme to translocate the DNA. Hemi-methylated DNA was the preferred substrate for methylation.

Restriction endonucleases R.EcoKI and R.EcoR124I are probably located in different environments within the bacterial cell.

We describe the phenomenon of a transient state of R124I restriction deficiency after long-term storage of the E. coli[pCP1005] strain at 4 degrees C, or after growth of the culture in synthetic M9 medium with the nonmutagenic solvent dimethyl sulfoxide. The unusual high reversion from the R+ 124 to the R- 124 phenotype was observed only in E. coli strain transformed with the high-copy number plasmid pCP1005 carrying EcoR124I hsdR, M and S genes cloned, but not with strains carrying the natural conjugative plasmid R124. The effect of both treatments on the expression of EcoR124I phenotype in relation to the possible location of R.EcoR124I restriction endonuclease in E. coli is discussed.

Overproduction of the Hsd subunits leads to the loss of temperature-sensitive restriction and modification phenotype.

The genes hsdM and hsdS for M. EcoKI modification methyltransferase and the complete set of hsdR, hsdM and hsdS genes coding for R. EcoKI restriction endonuclease, both with and without a temperature-sensitive (ts) mutation in hsdS gene, were cloned in pBR322 plasmid and introduced into E. coli C (a strain without a natural restriction-modification (R-M) system). The strains producing only the methyltransferase, or together with the endonuclease, were thus obtained. The hsdSts-1 mutation, mapped previously in the distal variable region of the hsdS gene with C1 245-T transition has no effect on the R-M phenotype expressed from cloned genes in bacteria grown at 42 degrees C. In clones transformed with the whole hsd region an alleviation of R-M functions was observed immediately after the transformation, but after subculture the transformants expressed the wild-type R-M phenotype irrespective of whether the wild-type or the 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.

Cloning, production and characterisation of wild type and mutant forms of the R.EcoK endonucleases.

The hsdR, hsdM and hsdS genes coding for R.EcoK restriction endonuclease, both with and without a temperature sensitive mutation (ts-1) in the hsdS gene, were cloned in pBR322 plasmid and introduced into E.coli C3-6. The presence of the hsdSts-1 mutation has no effect on the R-M phenotype of this construct in bacteria grown at 42 degrees C. However, DNA sequencing indicates that the mutation is still present on the pBR322-hsdts-1 operon. The putative temperature-sensitive endonuclease was purified from bacteria carrying this plasmid and the ability to cleave and methylate plasmid DNA was investigated. The mutant endonuclease was found to show temperature-sensitivity for restriction. Modification was dramatically reduced at both the permissive and non-permissive temperatures. The wild type enzyme was found to cleave circular DNA in a manner which strongly suggests that only one endonuclease molecule is required per cleavage event. Circular and linear DNA appear to be cleaved using different mechanisms, and cleavage of linear DNA may require a second endonuclease molecule. The subunit composition of the purified endonucleases was investigated and compared to the level of subunit production in minicells. There is no evidence that HsdR is prevented from assembling with HsdM and HsdSts-1 to produce the mutant endonuclease. The data also suggests that the level of HsdR subunit may be limiting within the cell. We suggest that an excess of HsdM and HsdS may produce the methylase in vivo and that assembly of the endonuclease may be dependent upon the prior production of this methylase.

Metabolism of diamantane by rat liver microsomal cytochromes P-450.

1. Diamantane binds to liver microsomes from phenobarbital-treated rats with an apparent Ks value of 5.2 x 10(-7) mol/l. This value being lower than that obtained for perhydrophenanthrene indicates that diamantane is very strongly bound to microsomal cytochrome P-450. 2. Metabolic studies show that liver microsomes from phenobarbital-treated rats readily metabolize diamantane to mono-, di- and possibly tri-hydroxy derivatives, whereas liver microsomes from beta-naphthoflavone-induced rats do not bind this hydrocarbon or metabolize it. 3. Reconstituted cytochromes P-450 b and e were more efficient in the hydroxylation of diamantane than liver microsomes; metabolites formed by the reconstituted system do not include all the products formed by microsomes, which indicates the involvement of forms of cytochrome P-450 other than the isozymes b and e.