Purification and Characterization of an w Protein from Micrococcus luteus*

An w protein capable of a concerted breaking and rejoining of DNA backbone bonds has been purified from Micrococcus luteus to homogeneity. The protein is a single polypeptide with a molecular weight of 120,000. It catalyzes the removal of superhelical turns from a highly negatively twisted DNA efficiently. The reaction requires Mg2+. In a medium containing 0.1 M K+, the enzyme acts in distributive fashion. It becomes more processive when the salt concentration is lowered. The rate of the reaction is strongly dependent on the sense and degree of superhelicity of the DNA substrate; with DNA substrates containing a few negative or positive superhelical turns the reaction is very slow. It appears that the prokaryotic w proteins isolated from M. Zuteus and Escherichia coli have rather similar enzymatic properties, and both differ from similar proteins from eukaryotic organisms in their Mg2+ requirement and in their strong dependence on superhelicity. The M. luteus enzyme also catalyzes the formation of knotted single-stranded DNA rings (Liu, L. F., Depew, R. E., and Wang, J. C. (1976) J. Mol. Biol. 106,439-452). TheM. luteus and E. coli o proteins are immunologically rather different however. Antibodies against one do not affect the activity of the other significantly.