Oligomeric state of the N-terminal domain of DnaT for replication restart in Escherichia coli.

Shogo Inoue, Yohei Ikeda, Saki Fujiyama, Tadashi Ueda, Yoshito Abe

Biochimica et biophysica acta. Proteins and proteomics 2023 Sep 01

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DNA replication stops when chemical or physical damage occurs to the DNA. Repairing genomic DNA and reloading the replication helicase are crucial steps for restarting DNA replication. The Escherichia coli primosome is a complex of proteins and DNA responsible for reloading the replication helicase DnaB. DnaT, a protein found in the primosome complex, contains two functional domains. The C-terminal domain (89-179) forms an oligomeric complex with single-stranded DNA. Although the N-terminal domain (1-88) forms an oligomer, the specific residues responsible for this oligomeric structure have not yet been identified. In this study, we proposed that the N-terminal domain of DnaT has a dimeric antitoxin structure based on its primary sequence. Based on the proposed model, we confirmed the site of oligomerization in the N-terminal domain of DnaT through site-directed mutagenesis. The molecular masses and thermodynamic stabilities of the site-directed mutants located at the dimer interface, namely Phe42, Tyr43, Leu50, Leu53, and Leu54, were found to be lower than those of the wild-type. Moreover, we observed a decrease in the molecular masses of the V10S and F35S mutants compared to the wild-type DnaT. NMR analysis of the V10S mutant revealed that the secondary structure of the N-terminal domain of DnaT was consistent with the proposed model. Additionally, we have demonstrated that the stability of the oligomer formed by the N-terminal domain of DnaT is crucial for its function. Based on these findings, we propose that the DnaT oligomer plays a role in replication restart in Escherichia coli. Copyright © 2023 Elsevier B.V. All rights reserved.

Citation

Shogo Inoue, Yohei Ikeda, Saki Fujiyama, Tadashi Ueda, Yoshito Abe. Oligomeric state of the N-terminal domain of DnaT for replication restart in Escherichia coli. Biochimica et biophysica acta. Proteins and proteomics. 2023 Sep 01;1871(5):140929