Role of centromere sites in activation of ParB proteins for partition complex assembly

Abstract

The ParB-parS partition complexes that bacterial replicons use to ensure their faithful inheritance also find employment in visualization of DNA loci, as less intrusive alternatives to fluorescent repressor-operator systems. The ability of ParB molecules to interact via their N-terminal domains and to bind to non-specific DNA enables expansion of the initial complex to a size both functional in partition and, via fusion to fluorescent peptides, visible by light microscopy. We have investigated whether it is possible to dispense with the need to insert parS in the genomic locus of interest, by determining whether ParB fused to proteins that bind specifically to natural DNA sequences can still assemble visible complexes. In yeast cells, coproduction of fusions of ParB to a fluorescent peptide and to a TALE protein targeting an endogenous sequence did not yield visible foci; nor did any of several variants of these components. In E.coli, coproduction of fusions of SopB (F plasmid ParB) to fluorescent peptide, and to dCas9 together with specific guide RNAs, likewise yielded no foci. The result of coproducing analogous fusions of SopB proteins with distinct binding specificities was also negative. We conclude that in order to assemble higher order partition complexes, ParB proteins need specific activation through binding to their cognate parS sites.

Rules and Exceptions: The Role of Chromosomal ParB in DNA Segregation and Other Cellular Processes

“…Nevertheless, no common assembly mechanism has been proposed, thus suggesting dynamic and heterogeneous interactions between ParB molecules within the complex (Figure 2b) [116,122,133,134]. It is widely acknowledged that ParB loading on parS is a prerequisite for the conformational changes that prime ParB for nucleation [135][136][137]. The ability of ParBs to build large nucleoprotein complexes may be a result of lateral ParB interactions (1D) and bridging interactions (3D) between ParB molecules located at distant DNA segments [117,134], clustering or building a ParB cage around parS by weak but dynamic interactions between protein dimers and DNA [129,138,139].…”