title: DnaT is a PriC-binding protein.
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.
authors.affiliation: School of Biomedical Sciences, Chung Shan Medical University, No.110, Sec.1, Chien-Kuo N. Rd., Taichung City, Taiwan.
authors.forename: Chien-Chih
authors.initials: CC
authors.lastname: Huang
authors.affiliation: School of Biomedical Sciences, Chung Shan Medical University, No.110, Sec.1, Chien-Kuo N. Rd., Taichung City, Taiwan; Department of Medical Research, Chung Shan Medical University Hospital, No.110, Sec.1, Chien-Kuo N. Rd., Taichung City, Taiwan. Electronic address: cyhuang@csmu.edu.tw.
authors.forename: Cheng-Yang
authors.initials: CY
authors.lastname: Huang
mesh_terms: D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000648:ultrastructure*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000648:ultrastructure*; D008956:Models, Chemical; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation
chemical_list: D004268:DNA-Binding Proteins; C104198:DnaT protein, E coli; D029968:Escherichia coli Proteins; C069995:PriC protein, E coli
keywords: DNA replication restart; DnaT; PriB; PriC; Primosome; SSB