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

Steff Horemans; Matthaios Pitoulias; Alexandria Holland; Emilie Pateau; Christophe Lechaplais; Dariy Ekaterina; Alain Perret; Panos Soultanas; Laurent Janniere

doi:10.1186/s12915-022-01278-3

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

Steff Horemans, Matthaios Pitoulias, Alexandria Holland, Emilie Pateau, Christophe Lechaplais, Dariy Ekaterina, Alain Perret, Panos Soultanas, Laurent Janniere

Department of Life Sciences

Research output: Contribution to journal › Article › peer-review

21 Citations (SciVal)

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.

Original language	English
Article number	87
Number of pages	25
Journal	BMC Biology
Volume	20
Issue number	1
Early online date	13 Apr 2022
DOIs	https://doi.org/10.1186/s12915-022-01278-3
Publication status	Published - 31 Dec 2022

Bibliographical note

Publisher Copyright:
© 2022, The Author(s).

Data Availability Statement

All data generated and analyzed in this study are available, either in this published
article or the supplementary material or in the MetaboLights repository
(https:// www. ebi. ac. uk/ metab oligh ts/ MTBLS 4415) [122]. Strains and plasmids
are available upon request from the authors as well as sequence details.

Acknowledgements

This work has benefited from the facilities and expertise of the France-Bio-
Imaging infrastructure supported by the Agence Nationale de la Recherche
(ANR-10-INSB-04, call “investissements d’Avenir”). Strains BTS8 and LAS26
were generous gifts from Lyle A. Simmons. Plasmid p2CT, pIC610, and
pMADΔspsABCDEF were gifts from James Berger, Dominique Le Coq and
Thomas Dubois, respectively. We thank Ioana Popescu for assistance with the
ClarioStar (BMG LABTECH, Fr), Olivier Espeli for help in cell fixation, and Dr
Jedd Bellamy-Carter for helping us with the mass spectrometry experiments
to assess the stoichiometry and stability of the PykA protein. We are grateful
to Dominique Le Coq, Nathalie Declercq, and Thomas Dubois for stimulating
discussions.

Funding

This work was supported by the Biotechnology Biological Sciences Research Council (BBSRC) grant (BB/R013357/1) to P.S., a BBSRC sub-contract (RIS 1165589) to L.J. and by recurring research funds from CNRS (Centre National de la Recherche Scientifique) and UEVE (Université d’Evry Val d’Essonne) to L.J.. S.H. was funded by a PhD fellowship from the MESR (Ministère de l’Enseignement Supérieur et de la Recherche; ED SDSV, Université Paris-Saclay, Université d’Evry Val d’Essonne). M.P. is a PhD student partially funded by a Vice Chancellor’s Excellence Award at the University of Nottingham. L.J. is on the CNRS staff. Funders had no role in the design of the study, the analysis and interpretation of data, and in the manuscript writing.

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

SDG 3 Good Health and Well-being

Keywords

Allosteric regulation
Cell cycle
Central carbon metabolism
Glycolytic enzymes
PEPut domain
Protein phosphorylation
Replication enzymes
Replication timing
Signaling

ASJC Scopus subject areas

Biotechnology
Structural Biology
Ecology, Evolution, Behavior and Systematics
Physiology
General Biochemistry,Genetics and Molecular Biology
General Agricultural and Biological Sciences
Plant Science
Developmental Biology
Cell Biology

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Cite this

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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.",

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T1 - Pyruvate kinase, a metabolic sensor powering glycolysis, drives the metabolic control of DNA replication

AU - Horemans, Steff

AU - Pitoulias, Matthaios

AU - Holland, Alexandria

AU - Pateau, Emilie

AU - Lechaplais, Christophe

AU - Ekaterina, Dariy

AU - Perret, Alain

AU - Soultanas, Panos

AU - Janniere, Laurent

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Y1 - 2022/12/31

N2 - 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.

AB - 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.

KW - Allosteric regulation

KW - Cell cycle

KW - Central carbon metabolism

KW - Glycolytic enzymes

KW - PEPut domain

KW - Protein phosphorylation

KW - Replication enzymes

KW - Replication timing

KW - Signaling

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U2 - 10.1186/s12915-022-01278-3

DO - 10.1186/s12915-022-01278-3

M3 - Article

SN - 1741-7007

VL - 20

JO - BMC Biology

JF - BMC Biology

IS - 1

M1 - 87

ER -

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

Abstract

Bibliographical note

Data Availability Statement

Acknowledgements

Funding

UN SDGs

Keywords

ASJC Scopus subject areas

Access to Document

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