Structural insights into the functional cycle of the Pseudomonas aeruginosa DnaB replicative helicase

The high-fidelity replication and inheritance of genetic information are fundamental to life. DNA helicases are essential molecular motors that unwind duplex DNA to provide single-stranded templates for replication and transcription. However, the molecular mechanism and structural basis of ATP-driven functional conformational dynamics exhibited by helicase remain poorly understood. Here, we report cryogenic electron microscopy structures of the Pseudomonas aeruginosa replicative helicase DnaB alone and in complex with its cognate primase DnaG, capturing 14 distinct states throughout its DNA unwinding cycle. Our integrative analysis reveals that ATP binding triggers concerted conformational and assembly remodeling, prompting the formation of higher-order oligomers, including heptamers and octamers. During unwinding, DnaB adopts a spiral staircase of H3-H4 motifs that translocates along single-stranded DNA. While multiple states coexist, their interconversion requires overcoming energy barriers. Dynamic interaction with primase DnaG stabilizes the N-terminal tier without further restricting the essential flexibility of the H3-H4 staircase. Our results reveal that ATP binding and hydrolysis facilitate processive translocation essentially by driving a conformational cycle of the H3-H4 staircase, rather than by directly powering strand separation. These findings provide a mechanistic framework for helicase function and a foundation for targeting this essential machine in a major bacterial pathogen.