Wozniak Katherine J, Burby Peter E, Nandakumar Jayakrishnan, Simmons Lyle A
Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, Michigan, United States of America.
PLoS Genet. 2022 May 16;18(5):e1010196. doi: 10.1371/journal.pgen.1010196. eCollection 2022 May.
CcrZ is a recently discovered cell cycle regulator that connects DNA replication initiation with cell division in pneumococci and may have a similar function in related bacteria. CcrZ is also annotated as a putative kinase, suggesting that CcrZ homologs could represent a novel family of bacterial kinase-dependent cell cycle regulators. Here, we investigate the CcrZ homolog in Bacillus subtilis and show that cells lacking ccrZ are sensitive to a broad range of DNA damage. We demonstrate that increased expression of ccrZ results in over-initiation of DNA replication. In addition, increased expression of CcrZ activates the DNA damage response. Using sensitivity to DNA damage as a proxy, we show that the negative regulator for replication initiation (yabA) and ccrZ function in the same pathway. We show that CcrZ interacts with replication initiation proteins DnaA and DnaB, further suggesting that CcrZ is important for replication timing. To understand how CcrZ functions, we solved the crystal structure bound to AMP-PNP to 2.6 Å resolution. The CcrZ structure most closely resembles choline kinases, consisting of a bilobal structure with a cleft between the two lobes for binding ATP and substrate. Inspection of the structure reveals a major restructuring of the substrate-binding site of CcrZ relative to the choline-binding pocket of choline kinases, consistent with our inability to detect activity with choline for this protein. Instead, CcrZ shows activity on D-ribose and 2-deoxy-D-ribose, indicating adaptation of the choline kinase fold in CcrZ to phosphorylate a novel substrate. We show that integrity of the kinase active site is required for ATPase activity in vitro and for function in vivo. This work provides structural, biochemical, and functional insight into a newly identified, and conserved group of bacterial kinases that regulate DNA replication initiation.
CcrZ是一种最近发现的细胞周期调节因子，它将肺炎链球菌中的DNA复制起始与细胞分裂联系起来，并且可能在相关细菌中具有类似功能。CcrZ也被注释为一种假定的激酶，这表明CcrZ同源物可能代表了一类新型的依赖激酶的细菌细胞周期调节因子家族。在这里，我们研究了枯草芽孢杆菌中的CcrZ同源物，并表明缺乏ccrZ的细胞对广泛的DNA损伤敏感。我们证明，ccrZ表达的增加会导致DNA复制过度起始。此外，CcrZ表达的增加会激活DNA损伤反应。以对DNA损伤的敏感性为指标，我们表明复制起始的负调节因子（yabA）和ccrZ在同一途径中发挥作用。我们表明CcrZ与复制起始蛋白DnaA和DnaB相互作用，进一步表明CcrZ对复制时间很重要。为了了解CcrZ的功能，我们解析了与AMP-PNP结合的CcrZ晶体结构，分辨率为2.6 Å。CcrZ结构与胆碱激酶最为相似，由一个双叶结构组成，两叶之间有一个裂缝用于结合ATP和底物。对该结构的检查揭示了CcrZ的底物结合位点相对于胆碱激酶的胆碱结合口袋有重大重组，这与我们无法检测到该蛋白对胆碱的活性一致。相反，CcrZ对D-核糖和2-脱氧-D-核糖有活性，表明CcrZ中胆碱激酶折叠结构已适应磷酸化一种新底物。我们表明，激酶活性位点的完整性对于体外ATP酶活性和体内功能是必需的。这项工作为新鉴定的、保守的调节DNA复制起始的细菌激酶组提供了结构、生化和功能方面的见解。
