Architecture of the ParF•ParG protein complex involved in prokaryotic DNA segregation

Abstract

SummaryThe mechanism by which low copy number plasmids are segregated at cell division involves the concerted action of two plasmid-encoded proteins that assemble on a centromere-like site. This study explores the topology of the DNA segregation machinery specified by the parFG locus of TP228, a partition system which is phylogenetically distinct from more well-characterized archetypes. A variety of genetic, biochemical and biophysical strategies revealed that the ParG protein is dimeric. ParF, which is more closely related to the cell division regulator MinD than to the prototypical ParA partition protein of plasmid P1, is instead multimeric and its polymeric state appears to be modulated by ATP which correlates with the proposed ATPbinding activity of ParF. ParG interacts in a sequencespecific manner with the DNA region upstream of the parFG locus and this binding is modulated by ParF. Intriguingly, the ParF and ParG proteins form at least two types of discrete complex in the absence of this region suggesting that the assembly dynamics of these proteins onto DNA is intricate.

The tail of the ParG DNA segregation protein remodels ParF polymers and enhances ATP hydrolysis via an arginine finger-like motif

“…1C). These observations exclude that the lack of stimulation of ParF ATPase activity by the ParG derivatives might be due to loss of interaction with ParF or perturbation of ParG tertiary structure; parF and parG alleles were cloned in pT18 and pT25, respectively, and analyzed as described (4). Colonies displaying an interaction are red, whereas, in the absence of interaction, they are white.…”

“…Assays were performed as described (4). ⌬9parG, ⌬19parG, and ⌬30parG were cloned in pT25 (35) as described (7).…”

“…His-tagged ParF, ParG, ⌬ParG, ParGR19K, and ParGR19A proteins were purified as described (4,7). Histagged ParF and ParG are as proficient as wild-type proteins in supporting plasmid partition in vivo (4).…”

“…ParG (8.6 kDa) is the prototype of a class of small proteins involved in accurate segregation that are unrelated phylogenetically to ParB, but that fulfil analogous functions as centromere-binding factors (1,4,5). ParG is dimeric, with symmetric C-terminal domains that interleave into a ribbon-helixhelix fold that is crucial for DNA binding, and unstructured N-terminal tails (4, 6).…”

“…ParG (8.6 kDa) is the prototype of a class of small proteins involved in accurate segregation that are unrelated phylogenetically to ParB, but that fulfil analogous functions as centromere-binding factors (1,4,5). ParG is dimeric, with symmetric C-terminal domains that interleave into a ribbon-helixhelix fold that is crucial for DNA binding, and unstructured N-terminal tails (4,6). Additional to its role as a centromerebinding protein, ParG is a transcriptional repressor of the parFG genes: transient associations between the flexible and folded domains in complex with target DNA modulate organization of a higher-order complex critical for transcriptional repression (7).…”