Kasho Kazutoshi, Miyoshi Kenya, Yoshida Mizuki, Sakai Ryuji, Nakagawa Sho, Katayama Tsutomu
Department of Molecular Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, Japan.
Nucleic Acids Res. 2025 Jan 11;53(2). doi: 10.1093/nar/gkae1291.
Oscillation of the active form of the initiator protein DnaA (ATP-DnaA) allows for the timely regulation for chromosome replication. After initiation, DnaA-bound ATP is hydrolyzed, producing inactive ADP-DnaA. For the next round of initiation, ADP-DnaA interacts with the chromosomal locus DARS2 bearing binding sites for DnaA, a DNA-bending protein IHF, and a transcription activator Fis. The IHF binding site is about equidistant between the DnaA and Fis binding sites within DARS2. The DARS2-IHF-Fis complex promotes ADP dissociation from DnaA and furnishes ATP-DnaA at the pre-initiation stage, which dissociates Fis in a negative-feedback manner. However, regulation for IHF binding as well as mechanistic roles of Fis and specific DNA structure at DARS2 remain largely unknown. We have discovered that negative DNA supercoiling of DARS2 is required for stimulating IHF binding and ADP dissociation from DnaA in vitro. Consistent with these, novobiocin, a DNA gyrase inhibitor, inhibits DARS2 function in vivo. Fis Gln68, an RNA polymerase-interaction site, is suggested to be required for interaction with DnaA and full DARS2 activation. Based on these and other results, we propose that DNA supercoiling activates DARS2 function by stimulating stable IHF binding and DNA loop formation, thereby directing specific Fis-DnaA interaction.
起始蛋白DnaA（ATP-DnaA）的活性形式的振荡允许对染色体复制进行适时调控。起始后，与DnaA结合的ATP被水解，产生无活性的ADP-DnaA。对于下一轮起始，ADP-DnaA与带有DnaA、一种DNA弯曲蛋白IHF和一种转录激活因子Fis结合位点的染色体位点DARS2相互作用。IHF结合位点在DARS2内的DnaA和Fis结合位点之间大致等距。DARS2-IHF-Fis复合物促进ADP从DnaA上解离，并在起始前阶段提供ATP-DnaA，ATP-DnaA以负反馈方式使Fis解离。然而，关于IHF结合的调控以及Fis的机制作用和DARS2处的特定DNA结构在很大程度上仍然未知。我们发现，DARS2的负超螺旋在体外刺激IHF结合和ADP从DnaA上解离是必需的。与此一致的是，DNA回旋酶抑制剂新生霉素在体内抑制DARS2功能。Fis Gln68是一个RNA聚合酶相互作用位点，被认为是与DnaA相互作用和DARS2完全激活所必需的。基于这些及其他结果，我们提出DNA超螺旋通过刺激稳定的IHF结合和DNA环形成来激活DARS2功能，从而指导特定的Fis-DnaA相互作用。
