CTP promotes efficient ParB-dependent DNA condensation by facilitating one-dimensional diffusion from parS
- Centro Nacional de Biotecnologia, Consejo Superior de Investigaciones Cientificas, Spain
- University of Bristol, United Kingdom
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 CTPgS. 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.
Data availability
All DNA sequences used are included in Supplementary Information.Data sets for Fig. 1E, 1F; Fig. 1-S1B, Fig.1-S1C; Fig.1-S4C, Fig1-S4D; Fig. 2C, 2G; Fig. 4C, 4D, 4E, 4F, 4G; Fig. 4-S1A, Fig. 4-S1B, Fig. 4-S1C; Fig. 5A, 5B; Fig. 6A, 6B, 6D, 6F, have been provided.
Article and author information
Author details
Mark Simon Dillingham
Fernando Moreno-Herrero
Funding
European Research Council (681299)
- Fernando Moreno-Herrero
Wellcome Trust (100401/Z/12/Z)
- Mark Simon Dillingham
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2021, Balaguer et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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CTP promotes efficient ParB-dependent DNA condensation by facilitating one-dimensional diffusion from parS
eLife 10:e67554.
https://doi.org/10.7554/eLife.67554
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