title: DnaT is a single-stranded DNA binding protein.
abstract: DnaT is one of the replication restart primosomal proteins required for reinitiating chromosomal DNA replication in bacteria. In this study, we identified and characterized the single-stranded DNA (ssDNA)-binding properties of DnaT using electrophoretic mobility shift analysis (EMSA), bioinformatic tools and two deletion mutant proteins, namely, DnaT26-179 and DnaT42-179. ConSurf analysis indicated that the N-terminal region of DnaT is highly variable. The analysis of purified DnaT and the deletion mutant protein DnaT42-179 by gel filtration chromatography showed a stable trimer in solution, indicating that the N-terminal region, amino acid 1-41, is not crucial for the oligomerization of DnaT. Contrary to PriB, which forms a single complex with a series of ssDNA homopolymers, DnaT, DnaT26-179 and DnaT42-179 form distinct complexes with ssDNA of different lengths and the size of binding site of 26 ± 2 nucleotides (nt). Using bioinformatic programs (ps)(2) and the analysis of the positively charged/hydrophobic residue distribution, as well as the biophysical results in this study, we propose a binding model for the DnaT trimer-ssDNA complex, in which 25-nt-long ssDNA is tethered on the surface groove located in the highly conserved C-terminal domain of DnaT. These results constitute the first study regarding ssDNA-binding activity of DnaT. Consequently, a hand-off mechanism for primosome assembly was modified.
authors.affiliation: School of Biomedical Sciences, Chung Shan Medical University, No. 110, Sec. 1, Chien-Kuo N. Rd, Taichung, Taiwan.
authors.forename: Yen-Hua
authors.initials: YH
authors.lastname: Huang
authors.forename: Min-Jon
authors.initials: MJ
authors.lastname: Lin
authors.forename: Cheng-Yang
authors.initials: CY
authors.lastname: Huang
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D004261:DNA Replication; D004269:DNA, Bacterial / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007711:Klebsiella pneumoniae / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins