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.
Highlights
Faithful chromosome segregation is essential in all domains of life if daughter cells are each to inherit the full set of genetic information
While reproducing a key result from Easter and Gober (2002) that showed Caulobacter crescentus ParA-ATP dissociated pre-bound ParB from palindromic single-stranded DNA fragment (parS) (Easter and Gober, 2002), we found that CTP could modulate the nucleation of Caulobacter ParB on parS
We noted that ParB + CTP slowly dissociated from parS even before the probe was returned to a protein-free buffer
Summary
Faithful chromosome segregation is essential in all domains of life if daughter cells are each to inherit the full set of genetic information. ParB nucleates on parS before spreading outwards to the flanking DNA and bridges/cages DNA together to form a nucleoprotein network in vivo (Breier and Grossman, 2007; Murray et al, 2006; Taylor et al, 2015; Graham et al, 2014; Sanchez et al, 2015; Debaugny et al, 2018; Broedersz et al, 2014; Funnell, 2016) This nucleoprotein complex recruits SMC to disentangle and organize replicated DNA (Gruber and Errington, 2009; Sullivan et al, 2009; Wang et al, 2017; Tran et al, 2017; Minnen et al, 2011). Despite the importance of spreading for proper chromosome segregation, the mechanism by which a few parS-bound ParB can recruit hundreds more ParB molecules to the vicinity of parS to assemble a high-molecular-weight nucleoprotein complex is not fully understood
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
