Unraveling the helicase loading mechanism in human pathogenic bacteria: a path towards DNA replication inhibition and new antimicrobial target discovery

DnaB replicative helicases are required to unwind DNA and expose single strands, to be used as template during the DNA replication. Its loading onto the origin of replication relies on replication factors termed helicase loaders. Most human pathogenic bacteria, including Mycobacterium tuberculosis (Mt) and Vibrio cholerae (Vc), possess the ancestral replicative helicase loader DciA. Despite the central role of this event, structural and kinetic details in DciA- dependent systems still need to be outlined. Here we shed new light on the helicase loading and activation pathway in Vc and Mt systems. Our cryo-EM structure of the ATPγS-bound VcDnaB•ssDNA complex displays features such that it represents an alternative translocation state than the previously determined GDP•AlF4bound DnaB, providing proofs on the nucleotide dependent conformational switching, reinforcing the proposed hand- over- hand model occurring during DNA unwinding. Then, the expulsion of DciA from the DnaB• ssDNA complex remains unknown. Our integrative biophysics approach proved that up to three copies of loader tightly interact with the helicase, leading to the suppression of its ATPase activity, thus controlling DnaB’s activation in the early stages of replication initiation. Alongside, thanks to a FRET-based platform, we measured the DciA-dependent DNA fork unwinding operated by DnaB, and then we turned to a specifically designed DNA bubble to capture the loading-dependent helicase translocation on a physiological substrate. Taken together, our data suggest that DciA loads DnaB onto ssDNA using the ringopening mechanism. In parallel, two unprecedented cryo-EM structures of the DnaA•DnaB•DciA complex together with a SAXS- and NMR-based structural investigation of the mycobacterial loader fulfill our work, aiming to sustain the convergent evolution theory and develop new antimicrobial tools.