The N-terminal domain of DnaT, a primosomal DNA replication protein, is crucial for PriB binding and self-trimerization.

Yen-Hua Huang, Cheng-Yang Huang

Biochemical and biophysical research communications 2013 Dec 13

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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. Copyright © 2013 Elsevier Inc. All rights reserved.

Citation

Yen-Hua Huang, Cheng-Yang Huang. The N-terminal domain of DnaT, a primosomal DNA replication protein, is crucial for PriB binding and self-trimerization. Biochemical and biophysical research communications. 2013 Dec 13;442(3-4):147-52