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ParB spreading on DNA requires cytidine triphosphate in vitro

View ORCID ProfileAdam S. B. Jalal, View ORCID ProfileNgat T. Tran, View ORCID ProfileTung B. K. Le
doi: https://doi.org/10.1101/2019.12.11.865972
Adam S. B. Jalal
1Department of Molecular Microbiology John Innes Centre, Norwich, NR4 7UH, United Kingdom
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Ngat T. Tran
1Department of Molecular Microbiology John Innes Centre, Norwich, NR4 7UH, United Kingdom
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Tung B. K. Le
1Department of Molecular Microbiology John Innes Centre, Norwich, NR4 7UH, United Kingdom
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  • For correspondence: tung.le@jic.ac.uk
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ABSTRACT

In all living organisms, it is essential to transmit genetic information faithfully to the next generation. The SMC-ParAB-parS system is widely employed for chromosome segregation in bacteria. A DNA-binding protein ParB nucleates on parS sites and must associate with neighboring DNA, a process known as spreading, to enable efficient chromosome segregation. Despite its importance, how the initial few ParB molecules nucleating at parS sites recruit hundreds of further ParB to spread is not fully understood. Here, we reconstitute a parS-dependent ParB spreading event using purified proteins from Caulobacter crescentus and show that CTP is required for spreading. We further show that ParB spreading requires a closed DNA substrate, and a DNA-binding transcriptional regulator can act as a roadblock to attenuate spreading unidirectionally in vitro. Our biochemical reconstitutions recapitulate many observed in vivo properties of ParB and opens up avenues to investigate the interactions between ParB-parS with ParA and SMC.

Footnotes

  • 1) Additional experiments: Figure1-figure supplement 1, Figure 3-figure supplement 2, Figure4-figure supplement 1, Figure 6, and Figure 6-figure supplement 2. 2) Updated Introduction, Materials and Methods, Results, and Discussion sections. 3) Mis-labelling from the original version: Morella thermoacetica His6-MBP-ParB (in the original manuscript) should be Dechloromonas aromatica His6-MBP-ParB, and 0.01 mM CTP was used for Zymomonas mobilis and Xanthomonas campestris ParB instead of 1mM CTP used for all other ParB samples. These two mislabellings have been corrected and the figure legend updated in the revised manuscript. The conclusion that ParB-CTP interaction with DNA is most likely conserved among ParB orthologs stands.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
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Posted February 20, 2020.
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ParB spreading on DNA requires cytidine triphosphate in vitro
Adam S. B. Jalal, Ngat T. Tran, Tung B. K. Le
bioRxiv 2019.12.11.865972; doi: https://doi.org/10.1101/2019.12.11.865972
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ParB spreading on DNA requires cytidine triphosphate in vitro
Adam S. B. Jalal, Ngat T. Tran, Tung B. K. Le
bioRxiv 2019.12.11.865972; doi: https://doi.org/10.1101/2019.12.11.865972

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