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

Chihiro Hayashi,Erika Miyazaki,Shogo Ozaki,Yoshito Abe,Tsutomu Katayama

doi:10.1074/jbc.ra120.014235

Chihiro Hayashi, Erika Miyazaki + Show 3 more

Open Access

https://doi.org/10.1074/jbc.ra120.014235

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Abstract

The DNA replication protein DnaA in Escherichia coli constructs higher-order complexes on the origin, oriC, to unwind this region. DnaB helicase is loaded onto unwound oriC via 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 unwound oriC via a weak interaction between DnaB and DnaA domain III. Previously, we showed that Phe46 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 on oriC but retained its DnaC-binding and helicase activities. DnaC binding moderately stimulated DnaA binding of DnaB L160A, and loading of DnaB L160A onto oriC was 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 impaired in vivo On the basis of these findings, we propose that DnaB Leu160 interacts with DnaA domain I Phe46 DnaB Leu160 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 onto oriC.

Highlights

Introduction ofWT dnaB on the plasmid enabled colony formation at 42 °C, whereas dnaB alleles bearing the L158A, V165A, or I168A mutation did not support growth (Table 1)
We performed NMR and mutant analyses of DnaA domain I, which showed that a patch containing Glu21 and Phe46 exposed on the surface of DnaA domain I binds to DnaB and that this interaction is essential for DnaB loading onto oriC [22, 23]
The results revealed that DnaB Leu160, located in the C terminus of DnaB N-terminal domain (NTD), was important for DnaA binding and DnaB loading onto oriC

Summary

Introduction

Introduction ofWT (wild-type) dnaB on the plasmid (pBAD/His-DnaB) enabled colony formation at 42 °C, whereas dnaB alleles bearing the L158A, V165A, or I168A mutation (pMS17, pMS18, or pMS19, respectively) did not support growth (Table 1). We performed NMR and mutant analyses of DnaA domain I, which showed that a patch containing Glu21 and Phe46 exposed on the surface of DnaA domain I binds to DnaB and that this interaction is essential for DnaB loading onto oriC [22, 23].

Results

Conclusion