Oligomeric state of the N-terminal domain of DnaT for replication restart in Escherichia coli |
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| Affiliation: | 1. Department of Protein Structure, Function and Design, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, Japan;2. Department of Pharmaceutical Sciences, School of Pharmacy at Fukuoka, International University of Health and Welfare, Okawa 831-8501, Japan;1. Department of Biomedical Engineering, Osaka Institute of Technology, Osaka 535-8585, Japan;2. Department of Applied Biological Science, Faculty of Agriculture, Kagawa University, Kagawa 761-0795, Japan;3. Division of Engineering, Faculty of Engineering, University of Fukui, Fukui 910-8507, Japan;1. Departamento de Bioquímica e Imunologia, Instituto Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil;2. Department of Veterinary Biomedical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, SK, Canada;3. Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY, USA;4. Laboratorio de Biología Molecular - Facultad de Ciencias Biológicas, Universidad Nacional Mayor de San Marcos (UNMSM), Peru;5. Diretoria de Pesquisa e Desenvolvimento, Fundação Ezequiel Dias, Belo Horizonte, Minas Gerais, Brazil;6. National Heart and Lung Institute, Faculty of Medicine, Imperial College London, Sir Alexander Fleming Building, London SW7 2AZ, UK;1. Program for the Study and Control of Tropical Diseases—PECET, Faculty of Medicine, University of Antioquia, Medellin, Colombia;2. Biophysics of Tropical Diseases, Max Planck Tandem Group, University of Antioquia, Medellin, Colombia;1. Institute of Biochemistry and Biophysics (IBB), University of Tehran, Tehran, Iran;2. Institute of Organic Chemistry, Albert-Ludwigs-University of Freiburg, Albertstrasse 21, Freiburg 79104, Germany;3. School of Life Sciences, Henan University, Kaifeng 475000, People''s Republic of China |
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| Abstract: | 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. |
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