Reversible autoinhibition of the bacterial replication-licensing Hda–β clamp complex

Abstract

Bacterial DNA replication must be tightly controlled to ensure once-per-cell-cycle initiation, requiring precise regulation of the initiator DnaA. The conserved RIDA pathway inactivates DnaA through the Hda–β sliding clamp (DnaN) complex, yet how this system is dynamically regulated has remained unclear. Here, we show that the Hda–DnaN complex exists as a concentration-dependent oligomeric ensemble rather than a static assembly. Using cryo-electron microscopy and mass photometry, we resolve multiple structural states, including an autoinhibited hetero-octamer, a hetero-hexamer intermediate, and a RIDA-compatible hetero-trimer. Structural comparison of these states suggests a plausible route of oligomeric interconversion involving progressive destabilization of Hda self-association, accompanied by exposure of the catalytic arginine finger required for DnaA inactivation. We further propose that this equilibrium operates as a titration-like mechanism that converts local protein concentration into functional states. Together, our findings establish reversible oligomerization as a potential regulatory principle for bacterial replication licensing.