Acta Microbiologica 27. (1980)

4. szám - Bánfalvi, G.–Ohlbaum, A.–Csuzi, S.–Antoni, F.: Purification and Characterization of a DNA-Dependent ATPase from Bacillus cereus

Acta microbiol. Acad. Sei. hung. 27, 289 — 297, 1980 PURIFICATION AND CHARACTERIZATION OF A DNA-DEPENDENT ATPase FROM BACILLUS CEREUS* G. Bánfalvi, Adél Ohlbaum, S. Csuzi and F. Antoni First Institute of Biochemistry, Semmelweis University Medical School, Budapest (Received June 29, 1979) A DNA-dependent ATPase (molecular weight 71 000) free of nuclease activity has been purified from Bacillus cereus. The enzyme shows similar characteristics as the enzyme isolated from Escherichia coli and Bacillus subtilis. Heat denatured DNA stimulates the rate of ATP hydrolysis to ADP and Pj to an extent about tenfold higher than the native DNA. Double stranded DNA without single stranded regions is not a suitable cofactor for the enzyme. The ATPase is inhibited by adenosine /i. y-imino)-diphosphate, while another ATP analogue, adenosine 5’-(/J, y-methylene)-diphosphate has no effect on ATPase activity. for ATP is 0.38 mM, the apparent К у, for nucleotide equivalent DNA is 1.2 рм. Evidence of the unwind, ing function of the enzyme is presented. DNA-dependent ATPases containing ATP-dependent DNase activity were described in various bacteria [1-5] and widely accepted as the enzymes of recombination. DNA-dependent ATPases without nucleolytic activity have been detected in Escherichia coli [6—9], Bacillus subtilis [10], vaccinia virus core [11], in mammalian [12, 13] and plant cells [14]. These ATPases stimulated by single stranded DNA may belong to the multienzyme system of DNA replication, unwinding double stranded DNA for the DNA synthesizing enzymes [15]. An ATP-dependent DNase having DNA stimulated ATPase activity was described earlier in B. cereus [5]. Some properties of this ATPase seemed to be similar to the DNA-dependent ATPases devoid of nuclease activity isolated from other bacteria [10, 16]. The question arose whether the DNA- dependent ATPase of B. cereus could be isolated in itself, separated from nuclease activity. The present paper describes the purification and main characteristics of a DNA-dependent ATPase from B. cereus, specific to single stranded DNA and free of DNase activity. Materials and methods Buffer A: 20 mM Tris-HCl pH 7.5, 0.1 mM ethylene-diamine tetraacetic acid, 2 mM 2-mercaptoethanol, 10% v/v glycerol. Buffer B: 20 mM Tris-HCl pH 7.5, 0.1 mM ethylene­­diamine-tetraacetic acid, 2 mM 2-mercaptoethanol, 30% v/v glycerol. Buffer C: 20 mM Tris­ * Work supported by the Hungarian Ministry of Health (4.01.5). Acta Microbiologica Academiae Scientiarum Hungaricae 27, 1980

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