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J. Biol. Chem., Vol. 281, Issue 8, 4606-4615, February 24, 2006

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DNA Reshaping by MukB RIGHT-HANDED KNOTTING, LEFT-HANDED SUPERCOILING^*

From the Department of Chemistry and Biochemistry, University of Oklahoma, Norman, Oklahoma 73019

MukB is a bacterial SMC (structural maintenance of chromosome) protein required for faithful chromosome segregation in Escherichia coli. We report here that purified MukB introduces right-handed knots into DNA in the presence of type-2 topoisomerase, indicating that the protein promotes intramolecular DNA condensation. The pattern of generated knots suggests that MukB, similar to eukaryotic condensins, stabilizes large right-handed DNA loops. In contrast to eukaryotic condensins, however, the net supercoiling stabilized by MukB was negative. Furthermore, DNA reshaping by MukB did not require ATP. These data establish that bacterial condensins alter the shape of double-stranded DNA in vitro and lend support to the notions that the right-handed knotting is the most conserved biochemical property of condensins. Finally, we found that MukB can be eluted from a heparin column in two distinct forms, one of which is inert in DNA binding or reshaping. Furthermore, we find that the activity of MukB is reversibly attenuated during chromatographic separation. Thus, MukB has a unique set of topological properties, compared with other SMC proteins, and is likely to exist in two different conformations.

Received for publication, April 29, 2005 , and in revised form, December 15, 2005.

^* This work was supported by National Institutes of Health Grant GM63786. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Chemistry and Biochemistry, University of Oklahoma, 620 Parrington Oval, Norman, OK 73019. Tel.: 405-325-1677; E-mail: valya{at}ou.edu.

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