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Nucleic Acids Research, 1993, Vol. 21, No. 3 373-379
© 1993

MOLECULAR BIOLOGY

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

Marie Weiserova, Pavel Janscak, Oldrich Benada, Josef Hubácek, Vitaly E. Zinkevich1, Stuart W. Glover1 and Keith Firman2,*

Laboratory of Molecular Genecs of Bacteria, Institute of Microbiology, Czechoslovak Academy of Sciences Videnska 1083, 142 20 Prague 4, Czechoslovakia 1Department of Biochemistry and Genetics, The Medical School, Catherine Cookson Building, The University Newcastle upon Tyne, NE2 4HH 2Biophysics Laboratories, School of Biological Sciences, University of Portsmouth St. Michael's Building, White Swan Road, Portsmouth P01 2DT, UK

*To whom correspondence should be addressed

Received November 27, 1992. Accepted December 15, 1992.

The hsdR, hsdM and hsdS genes coding for R·EcoK restriction endonuciease, both with and without a temperature sensitive mutation (ts-1) in the hsdS gene, were cloned in pBR322 plasmid and introduced into E.coll 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°C. However, DNA sequencing indicates that the mutation is still present on the pBR322-hsdts-1 operon. The putative temperaturesensitive endonuciease was purified from bacteria carrying this piasmid and the ability to cieave and methylate plasmid DNA was investigated. The mutant endonuclease was found to show temperaturesensitivity for restriction. Modification was dramaticaily reduced at both the permissive and non-permissive temperatures. The wiid type enzyme was found to cieave circuiar 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 miniceils. 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.


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