Negative DNA supercoiling enhances DARS2 binding of DNA-bending protein IHF in the activation of Fis-dependent ATP-DnaA production.

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.