Localization of the ParC protein in Escherichia coli cells

Abstract

The Escherichia coli ParC protein is one of two subunits of the topoisomerase IV, a type II topoisomerase. Topo IV is responsible for relaxing positive supercoils and decatenating linked daughter chromosomes after replication. This is done by introducing transient double-stranded breaks into the DNA, passing through a segment of uncut duplex DNA, before resealing the break. Mutations in parC lead to the par phenotype which is characterized by chromosomes that can replicate but are deficient in chromosomal partitioning. ParC is thus essential for cell survival. ParC has been found to associate with the replication machinery in the cell. ParE, Topo IV´s second subunit, was in the same study not found along with ParC. The difference in localization of ParC and ParE is proposed to underlie a temporal regulation of Topo IV´s activity in the cell. The activity of ParC may also be stimulated by SeqA, a protein preventing overinitiation of chromosome replication, and it is suggested that this stimulation is mediated by a specific interaction of Topo IV and SeqA.

In order to further look into ParC´s localization and function in the cell and to compare with SeqA´s assumed localization to the replication factory; both ParC and SeqA antibody were used in immunostaining followed by fluorescence microscopy. ParC antibody was, in opposition to SeqA antibody, not found to be localized as discrete foci and the co-localization of ParC protein to the replication factory is thus uncertain.

The ParC protein was purified in order to make an affinity column to purify the ParC antiserum used in the immunofluorescence microscopy. An expression plasmid was constructed by cloning the parC gene with a hexahistidine tag into a vector harbouring a T7 promoter. The expression plasmid was transformed into an E. coli strain that expresses T7 RNApolymerase. ParC protein was purified as a His-tagged protein by affinity chromatography based on the interaction between nickel and histidine, followed by gelfiltration.