论文标题
DNA复制启动和伸长率中的糖酵解丙酮酸激酶月光活性
Glycolytic pyruvate kinase moonlighting activities in DNA replication initiation and elongation
论文作者
论文摘要
细胞已经进化出对DNA复制的代谢控制，以应对广泛的营养条件。累积数据表明，至少部分取决于中央碳代谢（CCM），这种理解的控制尚不理解。在枯草芽孢杆菌中，糖酵解丙酮酸激酶（Pyka）与复制无关。这种585个氨基酸长的酶包括与磷酸烯醇丙酮酸（PEP）和ADP结合的催化（CAT）结构域，以产生丙酮酸和ATP，以及一个未知功能的C末端结构域。有趣的是，称为Peput的C末端结构域与CAT相互作用，并且是同源的域，在其他代谢酶中，该结构域以保守的TSH基序被磷酸化，以PEP和ATP为代价，以驱动糖进口，催化性或调节活性。为了洞悉Pyka在复制中的作用，在在Pyka的代谢活性以供生长的各种CAT和PEPUT突变体中分析了DNA合成。复制参数（ORI/TER比，C周期和叉子速度）和丙酮酸激酶活性的测量表明，Pyka突变体在Pyka蛋白中表现出复制缺陷，而不是由Pyka蛋白中的侧链修饰而不是其代谢活性的降低。有趣的是，CAT和PEPUT在复制方面具有不同的承诺：虽然CAT会对较正面和负复制的速度产生影响，但Peput却通过过程刺激启动，具体取决于猫型相互作用和生长条件。在CAT中与PEP和ADP结合的残基，稳定CAT-PEPUT相互作用并属于Peput的TSH基序，对于Pyka复制中的承诺很重要。在体外，Pyka影响复制酶的活性（聚合酶DNAE，解旋酶DNAC和Primase DNAG）对于启动和伸长以及与Pyka的基因相关的必不可少的。因此，我们的结果将复制起始和伸长率连接到CCM代谢产物（PEP，ATP和ADP），关键CAT和PEPUT残基，以及Pyka与Pyka与复制酶DNAE，DNAE，DNAC和DNAG之间的多个联系。我们建议Pyka具有月光活性，该活动感知信号代谢物的浓度，并与复制酶相互作用，以将有关细胞代谢状态的信息传达给复制机制，并调整复制起始和伸长率，以使代谢。这定义了一种新型的复制调节剂，该调节剂提议是在细胞周期中大门复制的代谢控制的一部分。
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.
