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.

Laboratory of Molecular Genetics of Bacteria Czechoslovak Academy of Sciences Prague

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Nucleic Acids Res 1993 Apr 11;21(7):1686 PubMed

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Gene. 1986;45(3):333-8 PubMed

J Mol Biol. 1981 Dec 5;153(2):425-40 PubMed

J Bacteriol. 1988 Apr;170(4):1775-82 PubMed

J Mol Biol. 1970 May 28;50(1):111-27 PubMed

Nature. 1970 Aug 15;227(5259):680-5 PubMed

Biochim Biophys Acta. 1973 Mar 19;299(2):177-88 PubMed

Fed Proc. 1974 May;33(5):1128-34 PubMed

J Biol Chem. 1972 Oct 10;247(19):6183-91 PubMed

Genetics. 1954 Jul;39(4):440-52 PubMed

Mol Gen Genet. 1980;180(1):35-46 PubMed

Mol Gen Genet. 1976 Jul 23;146(2):199-207 PubMed

Ann Inst Pasteur (Paris). 1954 Dec;87(6):653-73 PubMed

J Mol Biol. 1969 May 14;41(3):459-72 PubMed

J Bacteriol. 1953 Feb;65(2):113-21 PubMed

Annu Rev Biochem. 1972;41:447-66 PubMed

Mol Microbiol. 1987 Jul;1(1):13-22 PubMed

J Biochem Biophys Methods. 1990 Sep-Oct;21(3):247-66 PubMed

Cell. 1980 May;20(1):237-44 PubMed

Genetics. 1965 Nov;52(5):1043-50 PubMed

Methods Enzymol. 1979;68:493-503 PubMed

J Gen Microbiol. 1989 Nov;135(11):3057-65 PubMed

Mol Gen Genet. 1974;135(1):51-60 PubMed

J Mol Biol. 1979 May 15;130(2):191-209 PubMed

Gene. 1977;2(2):95-113 PubMed

J Mol Biol. 1992 Oct 5;227(3):597-601 PubMed

J Bacteriol. 1991 Oct;173(20):6568-77 PubMed

J Biol Chem. 1975 Jun 10;250(11):4159-64 PubMed

Gene. 1990 May 31;90(1):125-8 PubMed

J Mol Biol. 1983 May 5;166(1):1-19 PubMed

Dokl Akad Nauk SSSR. 1981;259(1):216-8 PubMed

Proc Natl Acad Sci U S A. 1988 Jul;85(13):4677-81 PubMed

Nucleic Acids Res. 1988 Feb 25;16(4):1563-75 PubMed

Proc Natl Acad Sci U S A. 1978 May;75(5):2271-5 PubMed

J Mol Biol. 1979 Apr 25;129(4):619-35 PubMed

J Gen Microbiol. 1980 Jul;119(1):231-8 PubMed

J Mol Biol. 1985 Nov 5;186(1):65-75 PubMed

Nature. 1968 Mar 23;217(5134):1110-4 PubMed

Curr Top Microbiol Immunol. 1984;108:1-9 PubMed

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