Restriction endonucleases and DNA methyltransferases constituting type-II restriction-modification (R-M) systems are well known for specific recognition of short nucleotide sequences (4-8 bp) within DNA. However, there is a striking difference between specificity of restriction enzymes and their cognate DNA methyltransferases. Studies on target recognition have shown that many restriction endonucleases cleave sites that differ by one base pair from their specific sites with rates reduced by factors of 100-1000. In case of cognate DNA methyltransferases such secondary sites are modified with rates only reduced by 1-2 orders of magnitude. It was also demonstrated for many restriction endonucleases, e.g. R.EcoRI, R.HindIII, as well as for methyltransferases, e.g. HaeIII that under some non-standard conditions when reaction mixtures were supplemented with dimethyl-sulfoxide (DMSO) or glycerol relaxed specificity (star activity) was easily detected. We are convinced that studies on relaxed specificity of R-M enzymes can open new venues to design enzymes with novel/tailored specificities.

Enzymes of the EcoVIII R-M system, isospecific to HindIII, recognize specific-sequence 5’-AAGCTT-3’/3’-TTCGAA-5’. Our studies concerns how such compounds like glycerol, ethanol, DMSO and Mn²⁺ affect recognizing non-canonical sequence by R.EcoVIII and M.EcoVIII and whether these effects are similar to those observed for R.HindIII. We have shown that 15% of DMSO and divalent cation Mn²⁺ induces star activity of R.HindIII. Our findings suggest that the same effect can be observed in case of ethanol at concentration of 10-20%. Similar results were obtained for EcoVIII restriction-modification enzymes. We have discovered that DMSO causes relaxation of specificity, which concerns both the examined enzymes. In case of R.EcoVIII this was within the range of 15-25% and for M.EcoVIII concentration of DMSO between 25 and 40% induced the relaxed specificity. Star activity of R.EcoVIII do not manifest itself in presence of glycerol and ethanol. However, it has been shown that 30-40% of glycerol provides to relaxed activity of M.EcoVIII.

It has been shown by others that under star-conditions R.HindIII recognises and digests secondary sequences (differing from canonical one-base pair) which appear to be: 5’-R↓AGCTT-3’, 5’-A↓GGCTT-3’, 5’-A↓TGCTT-3’, 5’-A↓AGCTT-3’, 5’-A↓ATCTT-3’, 5’-A↓AGCNT-3’, 5’-A↓AGCTY-3’ (where R=A or G; Y=C or T). Our findings reveal that for R.EcoVIII relaxed sequences are: 5’-C↓AGCTT-3’, 5’-A↓AGCTC-3’, 5’-A↓GGCTT-3’, 5’-C↓AGCTT-3’, 5’-A↓GGCTT-3’.

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1. Molecular analysis of the maintenance mechanism of plasmid pEC156 carrying EcoVIII restriction-modification system in bacteria related to Escherichia coli.