title: The N-terminal domain of DnaT, a primosomal DNA replication protein, is crucial for PriB binding and self-trimerization.
abstract: DnaT and PriB are replication restart primosomal proteins required for re-initiating chromosomal DNA replication in bacteria. Although the interaction of DnaT with PriB has been proposed, which region of DnaT is involved in PriB binding and self-trimerization remains unknown. In this study, we identified the N-terminal domain in DnaT (aa 1-83) that is important in PriB binding and self-trimerization but not in single-stranded DNA (ssDNA) binding. DnaT and the deletion mutant DnaT42-179 protein can bind to PriB according to native polyacrylamide gel electrophoresis, Western blot analysis, and pull-down assay, whereas DnaT84-179 cannot bind to PriB. In contrast to DnaT, DnaT26-179, and DnaT42-179 proteins, which form distinct complexes with ssDNA of different lengths, DnaT84-179 forms only a single complex with ssDNA. Analysis of DnaT84-179 protein by gel filtration chromatography showed a stable monomer in solution rather than a trimer, such as DnaT, DnaT26-179, and DnaT42-179 proteins. These results constitute a pioneering study of the domain definition of DnaT. Further research can directly focus on determining how DnaT binds to the PriA-PriB-DNA tricomplex in replication restart by the hand-off mechanism.
authors.affiliation: School of Biomedical Sciences, Chung Shan Medical University, No. 110, Sec. 1, Chien-Kuo N. Rd., Taichung City, Taiwan.
authors.forename: Yen-Hua
authors.initials: YH
authors.lastname: Huang
authors.forename: Cheng-Yang
authors.initials: CY
authors.lastname: Huang
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics*; D004261:DNA Replication; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D007711:Klebsiella pneumoniae / Q000235:genetics*; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D017384:Sequence Deletion
chemical_list: D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; C069996:PriB protein (N-terminal)
keywords: DNA replication restart; DnaT; EDTA; EMSA; IPTG; PriB; Primosome; SDS–PAGE; Single-stranded DNA; aa; amino acid residues; electrophoretic mobility shift analysis; ethylenediamine tetraacetic acid; isopropyl thiogalactoside; nt; nucleotides; single-stranded DNA; sodium dodecyl sulfate–polyacrylamide gel electrophoresis; ssDNA