糖酵解丙酮酸激酶在DNA复制起始和延伸中的兼职活性
Glycolytic pyruvate kinase moonlighting activities in DNA replication initiation and elongation
摘要：
细胞已经进化出对DNA复制的代谢控制，以响应广泛的营养条件。累积的数据表明，这种知之甚少的控制至少部分取决于中央碳代谢（CCM）。在枯草芽孢杆菌中，糖酵解丙酮酸激酶（PykA）与复制错综复杂地连接。这种585个氨基酸长的酶包含与磷酸烯醇丙酮酸（PEP）和ADP结合以产生丙酮酸和ATP的催化（Cat）结构域，以及未知功能的C末端结构域。有趣的是，被称为PEPut的C-末端结构域与Cat相互作用，并且是同源的结构域，在其他代谢酶中，该结构域以PEP和ATP为代价在保守的TSH基序处磷酸化，以驱动糖输入和催化或调节活性。为了深入了解PykA在复制中的作用，分析了在培养基中生长的各种Cat和PEPut突变体中的DNA合成，其中PykA的代谢活性被抑制生长。复制参数（ori/ter比，C期和叉速度）和丙酮酸激酶活性的测量表明，PykA突变体表现出复制缺陷，而不是从减少其代谢活性的PykA蛋白的侧链修饰。有趣的是，Cat和PEPut在复制方面有着不同的承诺：Cat对复制叉速度产生积极和消极的影响，而PEPut则通过一个依赖于Cat-PEPut相互作用和生长条件的过程来刺激启动。PykA在复制过程中与PEP和ADP结合的残基，稳定了Cat-PEPut相互作用，并且属于PEPut的TSH基序，对PykA的承诺很重要。在体外，PykA影响复制酶（聚合酶DnaE、解旋酶DnaC和引发酶DnaG）的活性，这些酶对起始和延伸至关重要，并与pykA遗传相关。因此，我们的研究结果将复制起始和延伸与CCM代谢产物（PEP，ATP和ADP），关键Cat和PEPut残基以及PykA与复制酶DnaE，DnaC和DnaG之间的多个链接联系起来。我们认为PykA具有兼职活性，可以感知信号代谢物的浓度，并与复制酶相互作用，将细胞代谢状态的信息传递给复制机制，并调整复制起始和延长代谢。这定义了一种新型的复制调节剂，被提议作为细胞周期中控制复制的代谢控制的一部分。
英文摘要：
Cells have evolved a metabolic control of DNA replication to respond to a wide range of nutritional conditions. Accumulating data suggest that this poorly understood control depends, at least in part, on Central Carbon Metabolism (CCM). In Bacillus subtilis , the glycolytic pyruvate kinase (PykA) is intricately linked to replication. This 585 amino-acid-long enzyme comprises a catalytic (Cat) domain that binds to phosphoenolpyruvate (PEP) and ADP to produce pyruvate and ATP, and a C-terminal domain of unknown function. Interestingly, the C-terminal domain termed PEPut interacts with Cat and is homologous a domain that, in other metabolic enzymes, are phosphorylated at a conserved TSH motif at the expense of PEP and ATP to drive sugar import and catalytic or regulatory activities. To gain insights into the role of PykA in replication, DNA synthesis was analyzed in various Cat and PEPut mutants grown in a medium where the metabolic activity of PykA is dispensable for growth. Measurements of replication parameters ( ori/ter ratio, C period and fork speed) and of the pyruvate kinase activity showed that PykA mutants exhibit replication defects resulting from side chain modifications in the PykA protein rather than from a reduction of its metabolic activity. Interestingly, Cat and PEPut have distinct commitments in replication: while Cat impacts positively and negatively replication fork speed, PEPut stimulates initiation through a process depending on Cat-PEPut interaction and growth conditions. Residues binding to PEP and ADP in Cat, stabilizing the Cat-PEPut interaction and belonging to the TSH motif of PEPut were found important for the commitment of PykA in replication. In vitro , PykA affects the activities of replication enzymes (the polymerase DnaE, helicase DnaC and primase DnaG) essential for initiation and elongation and genetically linked to pykA . Our results thus connect replication initiation and elongation to CCM metabolites (PEP, ATP and ADP), critical Cat and PEPut residues and to multiple links between PykA and the replication enzymes DnaE, DnaC and DnaG. We propose that PykA is endowed with a moonlighting activity that senses the concentration of signaling metabolites and interacts with replication enzymes to convey information on the cellular metabolic state to the replication machinery and adjust replication initiation and elongation to metabolism. This defines a new type of replication regulator proposed to be part of the metabolic control that gates replication in the cell cycle.
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