Pyruvate kinase, a metabolic sensor powering glycolysis, drives the metabolic control of DNA replication

Abstract

Background In all living organisms, DNA replication is exquisitely regulated in a wide range of growth conditions to achieve timely and accurate genome duplication prior to cell division. Failures in this regulation cause DNA damage with potentially disastrous consequences for cell viability and human health, including cancer. To cope with these threats, cells tightly control replication initiation using well-known mechanisms. They also couple DNA synthesis to nutrient richness and growth rate through a poorly understood process thought to involve central carbon metabolism. One such process may involve the cross-species conserved pyruvate kinase (PykA) which catalyzes the last reaction of glycolysis. Here we have investigated the role of PykA in regulating DNA replication in the model system Bacillus subtilis. Results On analysing mutants of the catalytic (Cat) and C-terminal (PEPut) domains of B. subtilis PykA we found replication phenotypes in conditions where PykA is dispensable for growth. These phenotypes are independent from the effect of mutations on PykA catalytic activity and are not associated with significant changes in the metabolome. PEPut operates as a nutrient-dependent inhibitor of initiation while Cat acts as a stimulator of replication fork speed. Disruption of either PEPut or Cat replication function dramatically impacted the cell cycle and replication timing even in cells fully proficient in known replication control functions. In vitro, PykA modulates activities of enzymes essential for replication initiation and elongation via functional interactions. Additional experiments showed that PEPut regulates PykA activity and that Cat and PEPut determinants important for PykA catalytic activity regulation are also important for PykA-driven replication functions. Conclusions We infer from our findings that PykA typifies a new family of cross-species replication control regulators that drive the metabolic control of replication through a mechanism involving regulatory determinants of PykA catalytic activity. As disruption of PykA replication functions causes dramatic replication defects, we suggest that dysfunctions in this new family of universal replication regulators may pave the path to genetic instability and carcinogenesis.

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

“…Particularly, mutants in pgm, pgk, eno, and pykA genes reversed the phenotypes of mutations in genes coding for DnaE (the lagging strand DNA polymerase), DnaC (the replicative helicase-the homologue of Escherichia coli DnaB), and DnaG (the primase), strongly suggesting the existence of a genetic connection between glycolysis and DNA replication. Subsequently, PykA was shown to exhibit an important regulatory role linking metabolism to replication [4], and recently, PykA has been shown to typify a novel family of cross-species replication regulators that drive the metabolic control of replication through Life 2023, 13, 965 2 of 17 a mechanism involving regulatory determinants of PykA catalytic activity [5]. Surprisingly, the disruption of this regulatory control causes dramatic replication and cell cycle defects, showing that the metabolic control of replication is important for the overall rate of DNA synthesis [5].…”

“…Subsequently, PykA was shown to exhibit an important regulatory role linking metabolism to replication [4], and recently, PykA has been shown to typify a novel family of cross-species replication regulators that drive the metabolic control of replication through Life 2023, 13, 965 2 of 17 a mechanism involving regulatory determinants of PykA catalytic activity [5]. Surprisingly, the disruption of this regulatory control causes dramatic replication and cell cycle defects, showing that the metabolic control of replication is important for the overall rate of DNA synthesis [5]. As failures in replication control increase the risk of replication errors and double-stranded DNA breaks, dysregulation of the metabolic control of replication may pave the way for genetic instability and carcinogenesis [6][7][8][9].…”

“…The PEPut domain is not required for the catalytic activity of PykA [16]. Instead, it has a regulatory role on the enzyme activity via its interaction with CAT and the phosphorylation status of its TSH motif [5]. PEPut interacts with CAT via a hydrogen bond between E209 and L536, assisting the heterotrophic effectors in stabilizing the active R-state conformation [15,29].…”

“…The CAT and PEPut domains were shown to exert different effects on DNA replication. CAT is a stimulator of elongation and PEPut is a nutrientdependent inhibitor of initiation [5]. These activities depend on substrates binding to CAT, the CAT-PEPut interaction, and the phosphorylation status of the TSH motif.…”

“…These activities depend on substrates binding to CAT, the CAT-PEPut interaction, and the phosphorylation status of the TSH motif. The direct functional effects of PykA on the activities of the DnaE and DnaC replication proteins were demonstrated with in vitro primer extension assays and helicase assays, respectively [5]. Despite the functional evidence of an interaction between PykA and the replication proteins DnaE and DnaC, there is no direct physical evidence of a protein-protein interaction.…”