Physical and functional interaction between a bacterial condensin subunit MukB and topoisomerase IV in Escherichia coli.

摘要

Proper geometric and topological organization of DNA is essential for all chromosomal processes. Two classes of proteins play major roles in organizing chromosomes: condensin complexes and type II topoisomerases. In Escherichia coli, MukB, an SMC-like component of the bacterial condensin, and topoisomerase IV (Topo IV), a type II topoisomerase that decatenates the newly replicated daughter chromosomes, are both essential for chromosome segregation in rapidly growing cells. However, little was known about the interplay between MukB and Topo IV. I demonstrated a physical and functional interaction between MukB and ParC, a subunit of Topo IV. The site of MukB-interaction was located on the C-terminal domain of ParC and a loss-of-interaction mutant, ParC R705E R729A, was isolated. This variant retained full activity as a topoisomerase when reconstituted with ParE to form Topo IV. Using mutant variants of ParC and MukB, we found that the MukB-ParC interaction enhanced relaxation of negatively supercoiled DNA and knotting by topoisomerase IV, which are intramolecular DNA rearrangements, but not decatenation of multiply-linked plasmid dimers, which is required for proper separation of daughter chromosomes. A specific chiral arrangement of the DNA was required for topoisomerase IV stimulation because relaxation of positively supercoiled DNA was unaffected. These data suggest that the MukB-ParC interaction plays a role in chromosome organization rather than in separation of daughter chromosomes. Consistent with this notion, introduction of loss-of-ParC-interaction variant mukB alleles to the cell caused abnormal nucleoid morphology distinct from a partition phenotype, which is emblematic of defects in chromosome segregation.