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

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

*Corresponding author for this work

Research output: Contribution to journalArticle (Academic Journal)peer-review

1 Citation (Scopus)
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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.

Original languageEnglish
Article numbere67554
JournaleLife
Volume10
DOIs
Publication statusPublished - 12 Jul 2021

Bibliographical note

Funding Information:
We are grateful to Michelle Hawkins (University of York) for supplying the EcoRI E111G variant. Work in the MSD lab was supported by the Wellcome Trust (New Investigator Award 100401/Z/12/Z to MSD) and the BBSRC (South West Biosciences Doctoral Training studentship to GLMF). FM-H acknowledges support from the European Research Council (ERC) under the European Union Horizon 2020 Research and Innovation Program (grant agreement 681299). Work in the Moreno-Herrero laboratory was also supported by Spanish Ministry of Economy and Competitiveness grant BFU2017-83794-P (AEI/FEDER, UE to FM-H) and Comunidad de Madrid grants Tec4-Bio – S2018/ NMT-4443 and NanoBioCancer – Y2018/BIO-4747 (to FM-H).

Publisher Copyright:
© Balaguer et al.

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