DNA reshaping by MukB. Right-handed knotting, left-handed supercoiling.
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.
Petrushenko
Zoya M
ZM
Department of Chemistry and Biochemistry, University of Oklahoma, Norman, Oklahoma 73019, USA.
Lai
Chien-Hung
CH
Rai
Rachna
R
Rybenkov
Valentin V
VV
D000213:Acyl Carrier Protein / Q000737:chemistry; D000251:Adenosine Triphosphatases / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D000266:Adenylyl Imidodiphosphate / Q000494:pharmacology; D002499:Centrifugation, Density Gradient; D002845:Chromatography; D002868:Chromosomal Proteins, Non-Histone / Q000737:chemistry / Q000502:physiology*; D004247:DNA / Q000737:chemistry*; D004269:DNA, Bacterial / Q000235:genetics; D004278:DNA, Superhelical / Q000737:chemistry*; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D015180:Electrophoresis, Gel, Two-Dimensional; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000502:physiology*; D006493:Heparin / Q000737:chemistry; D008854:Microscopy, Electron; D008954:Models, Biological; D008957:Models, Genetic; D008968:Molecular Conformation; D008970:Molecular Weight; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000378:metabolism; D012685:Sepharose / Q000494:pharmacology; D013395:Sucrose / Q000494:pharmacology
D000213:Acyl Carrier Protein; D002868:Chromosomal Proteins, Non-Histone; D004269:DNA, Bacterial; D004278:DNA, Superhelical; D029968:Escherichia coli Proteins; C067035:MukB protein, E coli; D000266:Adenylyl Imidodiphosphate; D013395:Sucrose; D000255:Adenosine Triphosphate; D006493:Heparin; D004247:DNA; D012685:Sepharose; D000251:Adenosine Triphosphatases