Molecular characterisation of a type III restriction-modification system in Campylobacter upsaliensis.

Two examples of Campylobacter upsaliensis RM3195 and JV21 strains are shown to carry putative type III restriction (res)-modification (mod) enzyme gene clusters, following genome sequence analyses. It is suggested that the cluster is composed of at least three structural genes, res, internal methylase gene and mod, in the strains, based on the nucleotide sequence information. A ribosome binding site, a putative promoter consisting of a consensus sequence at the -10-like structure and a semiconserved T-rich region and a putative intrinsic p-independent transcriptional terminator were identified for the gene cluster in the two strains. Using two primer pairs, f-/r-res and f-/r-mod, 34 of 41 C. upsaliensis isolates generated two expected amplicons of the res and mod gene segments, and using another primer pair, the same number of isolates also generated an amplicon of the res and mod gene segments cluster, including the third internal methylase gene. Thus, C. upsaliensis isolates frequently carried putative type III R-M gene clusters, encoding the three enzymes. Interestingly, two possible overlaps were identified within the three tandem structural genes. In addition, the type III R-M gene cluster loci appear to be very similar among the C. upsaliensis isolates and very different from other thermophilic campylobacters.

S-adenosyl-L-methionine is required for DNA cleavage by type III restriction enzymes.

The requirement of S-adenosyl-L-methionine (AdoMet) in the cleavage reaction carried out by type III restriction-modification enzymes has been investigated. We show that DNA restriction by EcoPI restriction enzyme does not take place in the absence of exogenously added AdoMet. Interestingly, the closely related EcoP15I enzyme has endogenously bound AdoMet and therefore does not require the addition of the cofactor for DNA cleavage. By employing a variety of AdoMet analogs, which differ structurally from AdoMet, this study demonstrates that the carboxyl group and any substitution at the epsilon carbon of methionine is absolutely essential for DNA cleavage. Such analogs could bring about the necessary conformational change(s) in the enzyme, which make the enzyme proficient in DNA cleavage. Our studies, which include native polyacrylamide gel electrophoresis, molecular size exclusion chromatography, UV, fluorescence and circular dichroism spectroscopy, clearly demonstrate that the holoenzyme and apoenzyme forms of EcoP15I restriction enzyme have different conformations. Furthermore, the Res and Mod subunits of the EcoP15I restriction enzyme can be separated by gel filtration chromatography in the presence of 2 M NaCl. Reconstitution experiments, which involve mixing of the isolated subunits, result in an apoenzyme form, which is restriction proficient in the presence of AdoMet. However, mixing the Res subunit with Mod subunit deficient in AdoMet binding does not result in a functional restriction enzyme. These observations are consistent with the fact that AdoMet is required for DNA cleavage. In vivo complementation of the defective mod allele with a wild-type mod allele showed that an active restriction enzyme could be formed. Furthermore, we show that while the purified c2-134 mutant restriction enzyme is unable to cleave DNA, the c2-440 mutant enzyme is able to cleave DNA albeit poorly. Taken together, these results suggest that AdoMet binding causes conformational changes in the restriction enzyme and is necessary to bring about DNA cleavage.

Type III restriction endonucleases translocate DNA in a reaction driven by recognition site-specific ATP hydrolysis.

Type III restriction/modification systems recognize short non-palindromic sequences, only one strand of which can be methylated. Replication of type III-modified DNA produces completely unmethylated recognition sites which, according to classical mechanisms of restriction, should be signals for restriction. We have shown previously that suicidal restriction by the type III enzyme EcoP15I is prevented if all the unmodified sites are in the same orientation: restriction by EcoP15I requires a pair of unmethylated, inversely oriented recognition sites. We have now addressed the molecular mechanism of site orientation-specific DNA restriction. EcoP15I is demonstrated to possess an intrinsic ATPase activity, the potential driving force of DNA translocation. The ATPase activity is uniquely recognition site-specific, but EcoP15I-modified sites also support the reaction. EcoP15I DNA restriction patterns are shown to be predetermined by the enzyme-to-site ratio, in that site-saturating enzyme levels elicit cleavage exclusively between the closest pair of head-to-head oriented sites. DNA restriction is blocked by Lac repressor bound in the intervening sequence between the two EcoP15I sites. These results rule out DNA looping and strongly suggest that cleavage is triggered by the close proximity of two convergently tracking EcoP15I-DNA complexes.

Interaction of EcoP1 modification methylase with S-adenosyl-L-methionine: a UV-crosslinking study.

EcoP1 modification methylase was radioactively labeled when incubated with S-adenosyl-L-[methyl-3H]methionine in the presence of ultraviolet light. Crosslinking of the enzyme as detected by electrophoresis on sodium dodecyl sulfate-polyacrylamide gel followed by fluorography and autoradiography, was shown to be specific by a number of criteria. More importantly, EcoP1 modification methylase was also radioactively labeled with S-adenosyl-L-[carboxyl-14C]methionine demonstrating that labeling involved binding of the entire AdoMet molecule rather than methylation of the protein. Further, c2 EcoP1 mutant DNA modification methylases which show negligible or very little methylation activity, correspondingly formed a weak or no adduct upon crosslinking. These results suggest that photolabeling of EcoP1 DNA modification methylase occurs at the AdoMet binding site.

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.