Abstract
Walker-box partition systems are ubiquitous in nature and mediate the segregation of bacterial and archaeal DNA. Well-studied plasmid Walker-box partition modules require ParA, centromere-DNA, and a centromere-binding protein, ParB. In these systems, ParA-ATP binds nucleoid DNA and uses it as a substratum to deliver ParB-attached cargo DNA, and ParB drives ParA dynamics, allowing ParA progression along the nucleoid. How ParA-ATP binds nonspecific DNA and is regulated by ParB is unclear. Also under debate is whether ParA polymerizes on DNA to mediate segregation. Here we describe structures of key ParA segregation complexes. The ParA-β,γ-imidoadenosine 5'-triphosphate (AMPPNP)-DNA structure revealed no polymers. Instead, ParA-AMPPNP dimerization creates a multifaceted DNA-binding surface, allowing it to preferentially bind high-density DNA regions (HDRs). DNA-bound ParA-AMPPNP adopts a dimer conformation distinct from the ATP sandwich dimer, optimized for DNA association. Our ParA-AMPPNP-ParB structure reveals that ParB binds at the ParA dimer interface, stabilizing the ATPase-competent ATP sandwich dimer, ultimately driving ParA DNA dissociation. Thus, the data indicate how harnessing a conformationally adaptive dimer can drive large-scale cargo movement without the requirement for polymers and suggest a segregation mechanism by which ParA-ATP dimers equilibrate to HDRs shown to be localized near cell poles of dividing chromosomes, thus mediating equipartition of attached ParB-DNA substrates.
Highlights
The propagation of genetic material, termed DNA segregation or partition, is essential for all life and is one of the most fundamental biological processes
The pNOB8 ParA protein is encoded on the pNOB8 plasmid, which is harbored in Sulfolobus NOB8H2 and is part of a partition cassette that encodes a centromere-binding protein (CBP) and adaptor (Schumacher et al 2015)
The largest buried surface area between dimers in the structure is between the adjacent DNA-bound dimers shown in Figure 1B (∼200 Å2) (Fig. 1C,D; Supplemental Fig. S2A,B), and only a few side chain interactions were observed between ParA molecules that surround a given DNA (Fig. 1C; Supplemental Fig. S2B)
Summary
The propagation of genetic material, termed DNA segregation or partition, is essential for all life and is one of the most fundamental biological processes. Two distinct mechanisms have been proposed for Walker-box partition: a polymer-based model in which ParA proteins form filaments on nsDNA that move and direct ParB– DNA cargo (Barillà et al 2005; Lim et al 2005; Ebersbach et al 2006; Bouet et al 2007; Hatano et al 2007; Ringgaard et al 2009; Gerdes et al 2010; Ptacin et al 2010) and a nonpolymer diffusion ratchet-like mechanism in which the destabilization of ParA DNA binding by ParB establishes a ParA–ATP gradient on the nucleoid that attracts ParB–DNA cargo (Vecchiarelli et al 2010, 2013a,b, 2014; Hwang et al 2013). In both models, ParB acts as a conduit for delivery of the cargo DNA and a ParA effector
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