Enterovirus 3C Proteases: Structure-based Discovery of Inhibitors

Abstract

DnaT and PriC are replication restart primosomal proteins required for re-initiating chromosomal DNA replication. DnaT is a component of the PriA-dependent primosome, while PriC belongs to the PriC-dependent primosome. Whether DnaT can interact with PriC is still unknown. In this study, we define a direct interaction between PriC, a key initiator protein in PriC-mediated DNA replication restart, and DnaT, a DnaB/C complex loader protein, from Klebsiella pneumoniae. In fluorescence titrations, PriC bound to single-stranded DNA with a binding-site size of approximately 9 nt. Gold nanoparticle assay showed that the solution of DnaT–PriC changed from red to purple, which indicated the protein–protein interactions due to gold nanoparticle aggregate. In addition, this DnaT–PriC complex could be co-purified by the heparin HP column. Surface plasmon resonance analysis showed that the Kd value of DnaT bound to PriC was 2.9 × 10−8 M. These results constitute a pioneering study of the DnaT–PriC interaction and present a putative link between the two independent replication restart pathways, namely, PriA- and PriC-dependent primosome assemblies. Further research can directly focus on determining how DnaT binds to the PriC–SSB–DNA tricomplex and regulates the PriC-dependent replication restart.