DnaT is a single-stranded DNA binding protein

作者：Huang Yen Hua; Lin Min Jon; Huang Cheng Yang^*

来源：Genes to Cells, 2013, 18(11): 1007-1019.

DOI：10.1111/gtc.12095

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.

• 出版日期2013-11