The Replicative DnaE Polymerase of Bacillus subtilis Recruits the Glycolytic Pyruvate Kinase (PykA) When Bound to Primed DNA Templates

Holland, Alexandria; Pitoulias, Matthaios; Soultanas, Panos; Janniere, Laurent

Abstract

The glycolytic enzyme PykA has been reported to drive the metabolic control of replication through a mechanism involving PykA moonlighting functions on the essential DnaE polymerase, the DnaC helicase and regulatory determinants of PykA catalytic activity in Bacillus subtilis. The mutants of this control suffer from critical replication and cell cycle defects, showing that the metabolic control of replication plays important functions in the overall rate of replication. Using biochemical approaches, we demonstrate here that PykA interacts with DnaE for modulating its activity when the replication enzyme is bound to a primed DNA template. This interaction is mediated by the CAT domain of PykA and possibly allosterically regulated by its PEPut domain, which also operates as a potent regulator of PykA catalytic activity. Furthermore, using fluorescence microscopy we show that the CAT and PEPut domains are important for the spatial localization of origins and replication forks, independently of their function in PykA catalytic activity. Collectively, our data suggest that the metabolic control of replication depends on the recruitment of PykA by DnaE at sites of DNA synthesis. This recruitment is likely highly dynamic, as DnaE is frequently recruited to and released from replication machineries to extend the several thousand RNA primers generated from replication initiation to termination. This implies that PykA and DnaE continuously associate and dissociate at replication machineries for ensuring a highly dynamic coordination of the replication rate with metabolism.

Citation

Holland, A., Pitoulias, M., Soultanas, P., & Janniere, L. (2023). The Replicative DnaE Polymerase of Bacillus subtilis Recruits the Glycolytic Pyruvate Kinase (PykA) When Bound to Primed DNA Templates. Life, 13(4), Article 965. https://doi.org/10.3390/life13040965

• Journal Article Type: Article
• Acceptance Date: Apr 5, 2023
• Online Publication Date: Apr 7, 2023
• Publication Date: 2023-04
• Deposit Date: Apr 14, 2023
• Publicly Available Date: Apr 18, 2023
• Journal: Life
• Electronic ISSN: 2075-1729
• Publisher: MDPI AG
• Peer Reviewed: Peer Reviewed
• Volume: 13
• Issue: 4
• Article Number: 965
• DOI: https://doi.org/10.3390/life13040965
• Keywords: Microbiology; metabolism; replication; PykA; DnaE; moonlighting activity
• Public URL: https://nottingham-repository.worktribe.com/output/19464432
• Publisher URL: https://www.mdpi.com/2075-1729/13/4/965