Molecular Biology Division, Bhabha Atomic Research Centre, Mumbai, India; Life Sciences, Homi Bhabha National Institute, Mumbai, India.
Molecular Biology Division, Bhabha Atomic Research Centre, Mumbai, India; Life Sciences, University of Mumbai, Mumbai, India.
J Biol Chem. 2021 Jan-Jun;296:100451. doi: 10.1016/j.jbc.2021.100451. Epub 2021 Feb 21.
Deinococcus radiodurans harbors a multipartite ploid genome system consisting of two chromosomes and two plasmids present in multiple copies. How these discrete genome elements are maintained and inherited is not well understood. PprA, a pleiotropic protein involved in radioresistance, has been characterized for its roles in DNA repair, genome segregation, and cell division in this bacterium. Here, we show that PprA regulates ploidy of chromosome I and II and inhibits the activity of drDnaA, the initiator protein in D. radiodurans. We found that pprA deletion resulted in an increased genomic content and ploidy of both the chromosomal elements. Expression of PprA in trans rescued the phenotypes of the pprA mutant. To understand the molecular mechanism underlying these phenotypes, we characterized drDnaA and drDnaB. As expected for an initiator protein, recombinant drDnaA showed sequence-specific interactions with the putative oriC sequence in chromosome I (oriCI). Both drDnaA and drDnaB showed ATPase activity, also typical of initiator proteins, but only drDnaB exhibited 5'→3' dsDNA helicase activity in vitro. drDnaA and drDnaB showed homotypic and heterotypic interactions with each other, which were perturbed by PprA. Interestingly, PprA has inhibited the ATPase activity of drDnaA but showed no effect on the activity of drDnaB. Regulation of chromosome copy number and inhibition of the initiator protein functions by PprA strongly suggest that it plays a role as a checkpoint regulator of the DNA replication initiation in D. radiodurans perhaps through its interaction with the replication initiation machinery.
耐辐射球菌拥有一个由两个染色体和两个质粒组成的多分体倍性基因组系统，这些离散的基因组元件是如何被维持和遗传的，目前还不是很清楚。多效蛋白 PprA 参与了耐辐射性，在该细菌中，它的作用已被确定为参与 DNA 修复、基因组分离和细胞分裂。在这里，我们表明 PprA 调节染色体 I 和 II 的倍性，并抑制 drDnaA 的活性，drDnaA 是耐辐射球菌中 DNA 复制的起始蛋白。我们发现 pprA 缺失导致染色体元件的基因组含量和倍性增加。PprA 的表达在转座子中挽救了 pprA 突变体的表型。为了理解这些表型的分子机制，我们对 drDnaA 和 drDnaB 进行了表征。与预期的起始蛋白一样，重组 drDnaA 与染色体 I 中的假定 oriC 序列（oriCI）表现出序列特异性相互作用。drDnaA 和 drDnaB 都表现出 ATP 酶活性，这也是起始蛋白的典型特征，但只有 drDnaB 在体外表现出 5'→3'双链 DNA 解旋酶活性。drDnaA 和 drDnaB 彼此表现出同型和异型相互作用，这些相互作用被 PprA 扰乱。有趣的是，PprA 抑制了 drDnaA 的 ATP 酶活性，但对 drDnaB 的活性没有影响。PprA 对染色体拷贝数的调节和对起始蛋白功能的抑制强烈表明，它可能通过与复制起始机制的相互作用，在耐辐射球菌中充当 DNA 复制起始的检查点调节剂。
