D na T is a single‐stranded DNA binding protein

D na T 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 (ss DNA )‐binding properties of D na T using electrophoretic mobility shift analysis ( EMSA ), bioinformatic tools and two deletion mutant proteins, namely, D na T 26‐179 and D na T 42‐179. C on S urf analysis indicated that the N ‐terminal region of D na T is highly variable. The analysis of purified D na T and the deletion mutant protein D na T 42‐179 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 D na T . Contrary to P ri B , which forms a single complex with a series of ss DNA homopolymers, D na T , D na T 26‐179 and D na T 42‐179 form distinct complexes with ss DNA 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 D na T trimer–ss DNA complex, in which 25‐nt‐long ss DNA is tethered on the surface groove located in the highly conserved C ‐terminal domain of D na T . These results constitute the first study regarding ss DNA ‐binding activity of D na T . Consequently, a hand‐off mechanism for primosome assembly was modified.