Cytidine triphosphate promotes efficient ParB-dependent DNA condensation by facilitating one-dimensional spreading fromparS

Abstract

Faithful segregation of bacterial chromosomes relies on the ParABS partitioning system and the SMC complex. In this work, we used single molecule techniques to investigate the role of cytidine triphosphate (CTP) binding and hydrolysis in the critical interaction between centromere-like parS DNA sequences and the ParB CTPase. Using a combined dual optical tweezers confocal microscope, we observe the specific interaction of ParB with parS directly. Binding around parS is enhanced 4-fold by the presence of CTP or the non-hydrolysable analogue CTPγS. However, ParB proteins are also detected at a lower density in distal non-specific regions of DNA. This requires the presence of a parS loading site and is prevented by roadblocks on DNA, consistent with one dimensional diffusion by a sliding clamp. Magnetic tweezers experiments show that the spreading activity, which has an absolute requirement for CTP binding but not hydrolysis, results in the condensation of parS-containing DNA molecules at low nanomolar protein concentrations. We propose a model in which ParB-CTP-Mg2+ complexes move along DNA following loading at parS sites and protein:protein interactions result in the localised condensation of DNA within ParB networks.

Plasmid partitioning driven by collective migration of ParA between nucleoid lobes

“…Over the past several decades, extensive research on the ParABS system has shed light on numerous aspects of its function and role in DNA segregation, most notably the in vitro reconstitutions of the systems of the F and P1 plasmids (27,28,53). Even so, surprises remained as evidenced by the recent discovery that many ParB proteins are CTPases that act like DNA clamps, loading at the parS sites and subsequently sliding along the DNA (9)(10)(11)(12)(13)(14)(15)(16). At the same time, techniques such as super resolution microscopy and chromatin immunoprecipitation (ChIP-Seq) have deepened our understanding of how ParA and ParB behave within cells (21,25,40,44,49,52,54).…”

“…Over the past several decades, extensive research on the ParABS system has shed light on numerous aspects of its function and role in DNA segregation, most notably the in vitro reconstitutions of the ParABS systems of the F and P1 plasmids (Hwang et al, 2013;Vecchiarelli et al, 2014bVecchiarelli et al, , 2013. Even so, surprises remained as evidenced by the recent discovery that many ParB proteins are CTPases that act like DNA clamps, loading at the parS sites and subsequently sliding along the DNA (Antar et al, 2021;De Asis Balaguer et al, 2021;Jalal et al, 2021Jalal et al, , 2020Osorio-Valeriano et al, 2021Soh et al, 2019;Taylor et al, 2021). At the same time, techniques such as super resolution microscopy and chromatin immunoprecipitation (ChIP-Seq) have deepened our understanding of how ParA and ParB behave within cells (Debaugny et al, 2018;Guilhas et al, 2020;Ietswaart et al, 2014;Le Gall et al, 2016;Lim et al, 2014;Sanchez et al, 2015;Surovtsev et al, 2016b).…”

“…ParB, which also dimerizes, binds to parS and spreads out along the DNA for several kilobases to form the nucleoprotein 'partition complex', visible using fluorescent microscopy (7,8). Very recently, it was found that the ParB of several species/plasmids is a CTPase that can entrap and slide along the DNA, potentially explaining the observed spreading (9)(10)(11)(12)(13)(14)(15)(16). ParB also stimulates the ATPase activity of ParA, generating a local depletion of ParA-ATP around the plasmid (17)(18)(19).…”

“…ParB, which also dimerizes, binds to parS and spreads out along the DNA for several kilobases to form the nucleoprotein 'partition complex', visible using fluorescent microscopy (Hirano et al, 1998;Rodionov et al, 1999). Very recently, it was found that the ParB of several species/plasmids is a CTPase that can entrap and slide along the DNA, potentially explaining the observed spreading (Antar et al, 2021;De Asis Balaguer et al, 2021;Jalal et al, 2021Jalal et al, , 2020Osorio-Valeriano et al, 2021Soh et al, 2019;Taylor et al, 2021). ParB also stimulates the ATPase activity of ParA, generating a local depletion of ParA-ATP around the plasmid (Hatano et al, 2007;Ringgaard et al, 2009;Vecchiarelli et al, 2014a).…”