4.6 Article

DnaB helicase is recruited to the replication initiation complex via binding of DnaA domain I to the lateral surface of the DnaB N-terminal domain

期刊

JOURNAL OF BIOLOGICAL CHEMISTRY
卷 295, 期 32, 页码 11131-11143

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ELSEVIER
DOI: 10.1074/jbc.RA120.014235

关键词

DNA helicase; DNA replication; protein complex; protein dynamics; protein-protein interaction; oriC; origin DNA; DnaA; DnaB; DNA helicase

资金

  1. Japan Society for the Promotion of Science (JSPS) KAKENHI [JP17H03656, JP20H03212]

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The DNA replication protein DnaA inEscherichia coliconstructs higher-order complexes on the origin,oriC, to unwind this region. DnaB helicase is loaded onto unwoundoriCvia interactions with the DnaC loader and the DnaA complex. The DnaB-DnaC complex is recruited to the DnaA complex via stable binding of DnaB to DnaA domain I. The DnaB-DnaC complex is then directed to unwoundoriCvia a weak interaction between DnaB and DnaA domain III. Previously, we showed that Phe(46)in DnaA domain I binds to DnaB. Here, we searched for the DnaA domain I-binding site in DnaB. The DnaB L160A variant was impaired in binding to DnaA complex onoriCbut retained its DnaC-binding and helicase activities. DnaC binding moderately stimulated DnaA binding of DnaB L160A, and loading of DnaB L160A ontooriCwas consistently and moderately inhibited. In a helicase assay with partly single-stranded DNA bearing a DnaA-binding site, DnaA stimulated DnaB loading, which was strongly inhibited in DnaB L160A even in the presence of DnaC. DnaB L160A was functionally impairedin vivo. On the basis of these findings, we propose that DnaB Leu(160)interacts with DnaA domain I Phe(46). DnaB Leu(160)is exposed on the lateral surface of the N-terminal domain, which can explain unobstructed interactions of DnaA domain I-bound DnaB with DnaC, DnaG primase, and DnaA domain III. We propose a probable structure for the DnaA-DnaB-DnaC complex, which could be relevant to the process of DnaB loading ontooriC.

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