CTP promotes efficient ParB-dependent DNA condensation by facilitating one-dimensional diffusion from parS.

Francisco De Asis Balaguer,Gemma Lm Fisher,Mark Simon Dillingham,Clara Aicart-Ramos,Eva M Martin-Cuevas,Cesar L Pastrana,Sara De Bragança,Fernando Moreno-Herrero

doi:10.7554/elife.67554

Francisco De Asis Balaguer, Gemma Lm Fisher + Show 6 more

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https://doi.org/10.7554/elife.67554

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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 optical tweezers confocal microscope, we observe the specific interaction of ParB with parS directly. Binding around parS is enhanced 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 DNA. This requires the presence of a parS loading site and is prevented by protein roadblocks, consistent with one-dimensional diffusion by a sliding clamp. ParB diffusion on non-specific DNA is corroborated by direct visualization and quantification of movement of individual quantum dot labelled ParB. 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.

Highlights

In bacterial cells, the separation of sister chromosomes is performed by the ParABS system and the SMC complex (Marbouty et al, 2015; Song and Loparo, 2015; Wang et al, 2014)
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
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

Summary

Introduction

The separation of sister chromosomes is performed by the ParABS system and the SMC complex (Marbouty et al, 2015; Song and Loparo, 2015; Wang et al, 2014). We and others have shown that ParB can self-associate to form networks which include specific binding to parS sequences and non-specific binding to distal DNA segments Overall, this results in the condensation and bridging of DNA at low forces (below 1 pN) (Fisher et al, 2017; Graham et al, 2014; Madariaga-Marcos et al, 2019, 2018; Taylor et al, 2015), and could explain how distant regions of DNA are bound by limited numbers of ParB proteins as shown in ChIP experiments.

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