ATP-regulated interactions between P1 ParA, ParB and non-specific DNA that are stabilized by the plasmid partition site, parS

Abstract

Localization of the P1 plasmid requires two proteins, ParA and ParB, which act on the plasmid partition site, parS. ParB is a site-specific DNA-binding protein and ParA is a Walker-type ATPase with non-specific DNA-binding activity. In vivo ParA binds the bacterial nucleoid and forms dynamic patterns that are governed by the ParB–parS partition complex on the plasmid. How these interactions drive plasmid movement and localization is not well understood. Here we have identified a large protein–DNA complex in vitro that requires ParA, ParB and ATP, and have characterized its assembly by sucrose gradient sedimentation and light scattering assays. ATP binding and hydrolysis mediated the assembly and disassembly of this complex, while ADP antagonized complex formation. The complex was not dependent on, but was stabilized by, parS. The properties indicate that ParA and ParB are binding and bridging multiple DNA molecules to create a large meshwork of protein–DNA molecules that involves both specific and non-specific DNA. We propose that this complex represents a dynamic adaptor complex between the plasmid and nucleoid, and further, that this interaction drives the redistribution of partition proteins and the plasmid over the nucleoid during partition.

Cell-free study of F plasmid partition provides evidence for cargo transport by a diffusion-ratchet mechanism

“…The bridging activity of SopA and SopB studied here has features similar to the DNA-bridging complexes described for P1 and pSM19035 of Streptococcus pyogenes (14,24). For all systems, DNA bridging required both proteins and ATP, and bridge disassembly was coupled to ParB-stimulated ATP hydrolysis.…”

“…For all systems, DNA bridging required both proteins and ATP, and bridge disassembly was coupled to ParB-stimulated ATP hydrolysis. For P1, the complex was proposed to bridge plasmid and nucleoid and was referred to as the "nucleoid-adaptor complex" (NAC) (24). For pSM19035, the bridging was proposed to play a role in plasmid pairing as well as in forming an NAC-like complex (14).…”

Dissection of the ATPase Active Site of P1 ParA Reveals Multiple Active Forms Essential for Plasmid Partition

“…The nonhydrolyzable analog ATP␥S supported assembly of larger and more stable complexes than those formed with ATP. From these observations, we concluded that NAC assembly and disassembly required ATP binding and hydrolysis, respectively (26).…”

“…, NAC-We next tested the ability of ParAK122R to support the formation of NAC, the large ParA-ParB-DNA com- plexes that can be detected in sucrose gradient sedimentation and light scattering assays (26). The sedimentation assay measures the change that protein binding has on the migration of DNA through a sucrose gradient.…”

“…This change is a key time-delay step that sets up the patterning in response to ParB. The interaction of ParB with ParA and DNA in vitro results in a large protein-DNA complex called NAC, for nucleoid adaptor complex (26). Assembly requires ATP binding and disassembly requires hydrolysis.…”