Dual Role of DNA in Regulating ATP Hydrolysis by the SopA Partition Protein

In bacteria, mitotic stability of plasmids and many chromosomes depends on replicon-specific systems, which comprise a centromere, a centromere-binding protein and an ATPase. Dynamic self-assembly of the ATPase appears to enable active partition of replicon copies into cell-halves, but for Walker-box partition ATPases the molecular mechanism is unknown. ATPase activity appears to be essential for this process. DNA and centromere-binding proteins are known to stimulate the ATPase activity but molecular details of the stimulation mechanism have not been reported. We have investigated the interactions which stimulate ATP hydrolysis by the SopA partition ATPase of plasmid F. By using SopA and SopB proteins deficient in DNA binding, we have found that the intrinsic ability of SopA to hydrolyze ATP requires direct DNA binding by SopA but not by SopB. Our results show that two independent interactions of SopA act in synergy to stimulate its ATPase. SopA must interact with (i) DNA, through its ATP-dependent nonspecific DNA binding domain and (ii) SopB, which we show here to provide an arginine-finger motif. In addition, the latter interaction stimulates ATPase maximally when SopB is part of the partition complex. Hence, our data demonstrate that DNA acts on SopA in two ways, directly as nonspecific DNA and through SopB as centromeric DNA, to fully activate SopA ATP hydrolysis.Faithful segregation of low copy number plasmids in bacteria depends on partition loci, named Par. Such loci are composed of two genes, generically termed parA and parB, encoding an ATPase and a DNA-binding protein, respectively, and a cis-acting centromeric site parS (reviewed in Ref. 1). These three essential elements are sufficient for the partition process. ParBs assemble on parS to form nucleoprotein structures called partition complexes (2–6). ParA ATPases are considered to be motors that direct displacement and positioning of partition complexes inside the cell.Partition systems have been classified into two major types, distinguished by the nature of their ATPase proteins (7). Type I is characterized by Walker box ATPases, which are specified by many plasmids and most bacterial chromosomes. In some (Type Ia) the nucleotide-binding P-loop is preceded by an N-terminal regulatory domain, in the others (Type Ib) it is not. Type II specifies actin-like ATPases and is present on relatively few plasmids. It is presently the best understood system at the molecular level (8–10). However, the underlying mechanism that drives partition still remains elusive for both systems. Our work aims at the understanding of an archetypal representative of Type Ia, namely SopABC of the Escherichia coli plasmid F.The several activities of Type Ia ParA proteins are regulated by binding of adenine nucleotides (11, 12), which induce conformational changes in the proteins (13, 14). In their apo and/or ADP-bound forms these proteins display site-specific DNA binding activity, recognizing their cognate promoters through their N-terminal domains. Such activity is involved in the autoregulation of par operon expression (15, 16). In the ATP-bound form, they specifically interact with cognate partition complexes through contact with ParB proteins. The ATP-bound form of type I ParAs spontaneously forms polymers, which appear as bundled filaments in electron micrographs (12, 17–19). The role of these filaments is not understood but they could be related to the rapid movement of partition complexes in the cell. In vivo, ParA proteins form dynamic assemblies that move back and forth in the cell if the cognate ParB protein and parS centromere are present (20–23). The link between this oscillatory behavior and the segregation of partition complexes is not clear. They both require the ATPase activity of ParA proteins but the role of ATP hydrolysis in the partition process is not understood.It has long been known that ParA partition proteins exhibit low intrinsic ATPase activity (24, 25). ATP hydrolysis is modestly stimulated by either DNA or the cognate ParB alone but is strongly activated (up to 35-fold) when both DNA and ParBs are present (12, 24, 25). The lack of major stimulation of ATPase by DNA in the absence of ParB proteins has been taken to mean that the DNA-bound form of ParB is the effective activator (26). However, incorporation of centromere sites in the DNA added to ParB did not increase stimulation of ATPase (24, 25), leaving doubts as to the role of the partition complex in ATPase activation.The mechanism by which ATP hydrolysis acts in the partition process is not known for type I systems. This is in marked contrast to actin-based partition ATPases whose ATPase activity is stimulated in growing filaments (8), where it provokes the rapid disassembly of filaments unless these are capped by the cognate partition complex (9). Therefore, for the type II partition system, ATP hydrolysis ensures discrimination between unproductive filaments that are rapidly disassembled and productive filaments that drive partition complexes to opposite ends of the cell. This dynamic instability, which ensures elongation of actin-like filaments only between two partition complexes to be segregated, thus provides regulation of the partition process.Recently, it has been shown that two members of the type I ParA family, Soj of Thermus thermophilus and SopA of plasmid F, bind nonspecific DNA in the presence of ATP (12, 26). Two studies revealed that this DNA binding activity is essential for partition (27, 28). Importantly, it has been shown that a SopA mutant deficient in DNA binding no longer stimulates ATP hydrolysis efficiently, suggesting that DNA could play a direct role in the regulation of the ATPase activity (28). This finding raises the issue of the interactions required for activation of the type I partition ATPase activity by cognate proteins and DNA.In this study, we have investigated the mechanism of activation of ATP hydrolysis by SopA. First, we have found that the formation of the F partition complex is required for strong stimulation of the SopA intrinsic ATPase activity. We have also found that the partition complex and DNA stimulate ATP hydrolysis independently but that these two independent interactions act in synergy to amplify SopA ATPase activity. Lastly, we have identified an arginine finger motif in SopB responsible for the stimulation of SopA ATPase activity.