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Structural and biochemical analyses of Caulobacter crescentus ParB reveal the role of its N-terminal domain in chromosome segregation

ABSTRACT: The tripartite ParA-ParB-parS complex ensures faithful chromosome segregation in the majority of bacterial species. ParB nucleates on the centromere-like parS site and spreads to neighboring DNA to form a network of protein-DNA complexes. This nucleoprotein network in turn interacts with ParA to partition the parS locus, hence the chromosome to each daughter cell. Here, we determine the co-crystal structure of the C-terminal domain truncated ParB-parS complex from Caulobacter crescentus, and show that its N-terminal domain is inherently flexible and adopts multiple different conformations. We propose that the flexibility of the N-terminal domain might facilitate the spreading of ParB on the chromosome. Next, using ChIP-seq we show that ParBs from different bacterial species exhibit variation in their intrinsic capability for spreading, and that the N-terminal domain rather than the C-terminal domain is the main determinant for the variation in spreading. Finally, we show that the C-terminal domain of Caulobacter ParB does not possess non-specific DNA-binding activity in vitro. Engineered ParB variants with enhanced non-specific DNA-binding activity condense DNA in vitro but do not spread further than a wild-type protein in vivo. Taken together, our results emphasize the central role of the N-terminal domain in ParB spreading and faithful chromosome segregation.

ORGANISM(S): Caulobacter vibrioides NA1000 Escherichia coli

PROVIDER: GSE134665 | GEO | 2020/07/22

REPOSITORIES: GEO

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A CTP-dependent gating mechanism enables ParB spreading on DNA

Project description:Proper chromosome segregation is essential in all living organisms. The ParA-ParB-parS system is widely employed for chromosome segregation in bacteria. Previously, we showed that Caulobacter crescentus ParB requires cytidine triphosphate to escape the nucleation site parS to spread by sliding to the neighboring DNA. Here, we provide the structural basis for this transition from nucleation to spreading by solving co-crystal structures of a C-terminal domain truncated C. crescentus ParB with parS and with a CTP analog. Nucleating ParB is an open clamp, in which parS is captured at the DNA-binding domain (the DNA-gate). Upon binding CTP, the N-terminal domain (NTD) self-dimerizes to close the NTD-gate of the clamp. The DNA-gate also closes, thus driving parS into a compartment between the DNA-gate and the C-terminal domain. CTP hydrolysis and/or the release of hydrolytic products may re-open the gates. Overall, we suggest a CTP-operated gating mechanism that regulates ParB nucleation and spreading.

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