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

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.

研究背景：所有活体细胞中，DNA复制（DNA replication）在多样生长条件下均受到精密调控，以确保细胞分裂（cell division）前基因组能够按时且精准地完成复制。该调控过程的异常会引发DNA损伤（DNA damage），对细胞存活与人类健康（包括癌症（cancer）发生）造成潜在灾难性后果。为应对此类威胁，细胞通过已知的多种机制严格控制复制起始（replication initiation）。此外，细胞还通过一种尚未明确的过程，将DNA合成与营养丰度（nutrient richness）及生长速率（growth rate）相偶联，该过程被认为涉及中心碳代谢（central carbon metabolism）。其中一类相关过程可能涉及跨物种保守的丙酮酸激酶（pyruvate kinase, PykA），该酶催化糖酵解（glycolysis）的最后一步反应。本研究以模式系统（model system）枯草芽孢杆菌（Bacillus subtilis）为研究对象，探究了PykA在DNA复制调控中的作用。
研究结果：通过分析枯草芽孢杆菌PykA的催化（Cat）结构域与C端（PEPut）结构域的突变体，我们在PykA对细胞生长非必需的培养条件下观察到了复制表型（replication phenotypes）。此类表型与突变对PykA催化活性的影响无关，且未伴随代谢组（metabolome）的显著改变。PEPut结构域作为营养依赖性的复制起始抑制剂发挥功能，而Cat结构域则可促进复制叉的移动速度。即便在已知复制调控功能完全正常的细胞中，破坏PEPut或Cat的复制调控功能，也会显著影响细胞周期（cell cycle）与复制时序（replication timing）。体外（in vitro）实验表明，PykA可通过功能相互作用（functional interactions）调控复制起始与延伸（replication elongation）过程所必需的酶活性。额外实验结果表明，PEPut可调控PykA的催化活性，且参与调控PykA催化活性的Cat与PEPut结构域决定簇，同样对PykA介导的复制功能至关重要。
研究结论：基于本研究结果，我们推断PykA代表了一类全新的跨物种复制调控因子家族，该家族通过依赖PykA催化活性调控决定簇的机制，实现复制过程的代谢调控。由于破坏PykA的复制调控功能会引发严重的复制缺陷，我们推测这类新型通用复制调控因子的功能异常，可能会导致基因组不稳定（genetic instability）并最终诱发癌症发生。