Dynamics of recruitment of macromolecular complexes active on the mycobacterial orisome: mechanistic details and target validation.

Tuberculosis (TB) disease is a major global health threat. Mycobacterium tuberculosis (MTB), the causative agent of TB, is one of the most widespread bacterial pathogens and it causes ill-health among millions of people and 1.4 million deaths each year. Among the different aspects of MTB biology, the biochemical processes underlying DNA replication are essential steps of the bacterial infection cycle. Indeed, at various stages throughout the disease cycle, DNA replication pathways are essential for preserving the genome integrity and viability of bacilli located in pulmonary lesions. Key studies provided new insights into the dynamics of MTB replisome as well as on its hierarchical organization, that is functional to the faithful duplication of genetic material. However, MTB replisome remains relatively poorly characterized, and the working model of the multi-step DNA replication cascade is therefore inferred through comparison with model organisms. In canonical models, the triggering event of new DNA synthesis consists in the recognition of the origin of replication (oriC) by the DNA initiator protein (DnaA) that leads to the unwinding of the adjacent AT-rich region and to the formation of the so-called orisome, while the single-stranded regions are exposed to recruit the bacterial replicative helicase (DnaB). Despite the absence of a canonical helicase loader, it was recently demonstrated that MTB possesses an ancestral bacterial replicative operator named DciA, which displays the features of the replicative helicase-operating proteins associated with replication initiation. Even though the loading of the replicative helicase onto DNA is a key step of DNA synthesis, structural and kinetic details of this process in mycobacteria have so far not been investigated. Furthermore, experimental evidences support the role of DciA in coordinating the assembly of an unprecedented ternary complex that includes the initiator protein DnaA and the replicative helicase DnaB. Using an integrated structural biology and biochemistry approach this proposal aims to characterize the architecture and the dynamics of the DciA/DnaB and DnaA/DnaB/DciA complexes, in order to define the molecular details of the most important steps of DNA replication initiation in MTB. Moreover, as promising translational outcome in the context of drug discovery programs, the structural biology investigations will drive the design of peptide inhibitors interfering with the formation of such macromolecular complexes providing new insights for the design and development of active compounds for a novel pharmacological approach against TB.