Prokaryotic DNA segregation most commonly involves members of the Walker-type ParA superfamily. Here we show that the ParF partition protein specified by the TP228 plasmid is a ParA ATPase that assembles into extensive filaments in vitro. Polymerization is potentiated by ATP binding and does not require nucleotide hydrolysis. Analysis of mutations in conserved residues of the Walker A motif established a functional coupling between filament dynamics and DNA partitioning. The partner partition protein ParG plays two separable roles in the ParF polymerization process. ParF is unrelated to prokaryotic polymerizing proteins of the actin or tubulin families, but is a homologue of the MinD cell division protein, which also assembles into filaments. The ultrastructures of the ParF and MinD polymers are remarkably similar. This points to an evolutionary parallel between DNA segregation and cytokinesis in prokaryotic cells, and reveals a potential molecular mechanism for plasmid and chromosome segregation mediated by the ubiquitous ParA-type proteins.

The genomes of unicellular and multicellular organisms must be partitioned equitably in coordination with cytokinesis to ensure faithful transmission of duplicated genetic material to daughter cells. Bacteria use sophisticated molecular mechanisms to guarantee accurate segregation of both plasmids and chromosomes at cell division. Plasmid segregation is most commonly mediated by a Walker-type ATPase and one of many DNA-binding proteins that assemble on a cis-acting centromere to form a nucleoprotein complex (the segrosome) that mediates intracellular plasmid transport. Bacterial chromosome segregation involves a multipartite strategy in which several discrete protein complexes potentially participate. Shedding light on the basis of genome segregation in bacteria could indicate new strategies aimed at combating pathogenic and antibiotic-resistant bacteria.

ParA superfamily ͉ polymerization ͉ plasmid partition ͉ ATPase T he precise distribution of newly replicated genomes to progeny cells is imperative for stable transmission of genetic information. In bacteria, the most well characterized segregation mechanisms are specified by low-copy-number plasmids. These systems most frequently comprise two plasmid-encoded proteins, often termed ParA and ParB, that assemble on a cis-acting centromeric site. ParB directly binds the centromere, whereas ParA is recruited by interactions with ParB. The resulting segrosome complex is a positioning apparatus that localizes the attached plasmids to specific subcellular addresses (1, 2).The segregation locus of multidrug-resistance plasmid TP228 in Escherichia coli consists of the parF and parG genes and nearby parH centromere (3). 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).The ParA superfamily of ATPases, widely encoded by both chromosomes and plasmids, is characterized by a variant Walkertype ATP-binding motif (8). ParF (22.0 kDa) epitomizes one clade of the superfamily (3). In common with other ParA proteins, ParF is a weak ATPase whose nucleotide hydrolysis is enhanced Ϸ30-fold by ParG (9). ATP binding and/or hydrolysis by ParA proteins has long been recognized as a crucial facet of the segregation process, although its mechanistic purpose was uncertain (10-12). We have recently shown that ATP binding stimulates the polymerization of ParF into extensive multistranded filaments, whereas ADP antagonizes filamentation. ParG is another key modulator of polymerization (9). Mutagenesis of the ATP-binding site in Parf perturbed DNA segregation in vivo, ATP hydrolysis, and polymerization. We envisage that segrosome formation is initiated by site-specific binding of ParG to parH, generating paired complexes of specific topology. ParF is then recruited. ParF polymerization within the complex is controlled by nucleotide binding, by ParG-mediated stimulation of ATP hydrolysis, by remodeling effects of ParG, and, more speculatively, by cell cycle signals. Polymerization, or depolymerization, invokes separation of paired plasmids and their segregation in opposite poleward directions (1, 9).Arginine fingers stimulate nucleotide hydrolysis by NTPases through the action of an arginine side chain inserted into the catalytic niche (13,14). The arginine stabilizes the transition state through neutralization of negative charge...

The carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II plays an important role in transcription and processing of the nascent transcript by interacting with both transcription and RNA processing factors. We show here that the cleavage͞ polyadenylation factor IA of Saccharomyces cerevisiae directly contacts CTD. First by affinity chromatography experiments with yeast extracts we demonstrate that the Rna15p, Rna14p, and Pcf11p subunits of this complex are associated with phosphorylated CTD. This interaction is confirmed for Rna15p by yeast two-hybrid analysis. Second, Pcf11p, but not Rna15p, is shown to directly contact phosphorylated CTD based on in vitro binding studies with recombinant proteins. These findings establish a direct interaction of cleavage͞polyadenylation factor IA with the CTD. Furthermore, a quantitative analysis of transcription run-on performed on temperature-sensitive mutant strains reveals that the lack of either functional Rna14p or Pcf11p affects transcription termination more severely than the absence of a functional Rna15p. Moreover, these data reinforce the concept that CTD phosphorylation acts as a regulatory mechanism in the maturation of the primary transcript. In Saccharomyces cerevisiae the largest subunit of RNA polymerase II (pol II) contains a carboxyl-terminal domain (CTD) consisting of 26 tandem repeats of a heptapeptide module with the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser (1, 2). Two serines per heptad repeat may undergo reversible phosphorylation during each transcription cycle (3). The phosphorylation status of the CTD is correlated with different stages of the transcription cycle. Thus hypophosphorylated polymerase (IIA) is competent for initiation, whereas hyperphosphorylated polymerase (II0) is associated with transcription elongation. Increasing evidence has been provided in the last few years showing that the CTD acts as a direct physical link between transcription and nascent RNA processing: in mammals, the cleavage-polyadenylation specificity factor (CPSF) and the cleavage stimulation factor (CstF), as well as splicing factors and 5Ј capping enzyme, all bind to the CTD (4-9). Furthermore, CPSF and CstF copurify with pol II (4). The concept of a factor recruiting͞docking platform has emerged as one likely function of this peculiar polypeptide. Evidence for a more direct role of the CTD in polyadenylation has also been indicated (10).In S. cerevisiae, the proteins so far known to bind to the CTD are the Mediator complex (11, 12), the Elongator complex (13), proteins involved in 5Ј end capping of nascent RNA (8,14), and the Ess1 peptidylprolyl isomerase (15, 16). To date the interaction of cleavage͞polyadenylation factors with the CTD remains largely uncharacterized. In yeast 3Ј-end processing of pre-mRNA requires cleavage factor IA (CF IA), cleavage factor IB (CF IB), cleavage factor II (CF II), polyadenylation factor I (PFI), poly(A) polymerase, and the poly(A)-binding protein 1 (17-22). CF IA, which is involved in both cleavage and po...

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.

b -strand which forms part of the DNA recognition motif.

Although recent studies have provided a wealth of information about archaeal biology, nothing is known about the molecular basis of DNA segregation in these organisms. Here we unveil the machinery and assembly mechanism of the archaeal Sulfolobus pNOB8 partition system. This system employs three proteins; ParA, an atypical ParB adaptor and a centromere-binding component, AspA. AspA utilizes a spreading mechanism to create a DNA superhelix onto which ParB assembles. This supercomplex links to the ParA motor, which contains a bacteria-like Walker motif. The ParB C-domain harbors structural similarity to CenpA, which dictates eukaryotic segregation. Thus, this archaeal system combines bacteria-like and eukarya-like components, suggesting the possible conservation of DNA segregation principles across the three domains of life.

Background: ParF and ParG mediate TP228 plasmid segregation. Results: ATP binding to ParF activates segregation, and ADP binding to ParF antagonizes its segregation function. ParF-ADP is monomeric, and ParF-ATP is dimeric. ParF dimers assemble into polymers. Conclusion: ParF-ATP dimers serve as building blocks for polymer assembly. Significance: ParF-ATP polymers provide a mechanism for plasmid segregation.