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

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 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 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 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 onto oriC.

Concerted actions of DnaA complexes with DNA-unwinding sequences within and flanking replication origin oriC promote DnaB helicase loading

“…We revealed that a patch including Glu21 and Phe46 of DnaA domain I constitutes the primary DnaB-binding site ( 28 , 30 ). DnaB Leu160, located on the lateral surface of this helicase, was suggested to interact with DnaA Phe46 ( 7 ). In addition, our previous pull-down experiments suggest that the DnaA complexes constructed on the FL-DOR bind two DnaB helicases, whereas those on the Left-DOR bind only a single DnaB helicase ( 8 ).…”

“…Upon this oriC unwinding, two hexamers of DnaB helicases are bidirectionally loaded onto the resultant single-stranded (ss) region with the help of the DnaC helicase loader ( Fig. 1 B ), leading to bidirectional chromosomal replication ( 5 , 6 , 7 , 8 ). However, the fundamental mechanism underlying oriC -dependent bidirectional DnaB loading remains elusive.…”

“…Domain I supports weak domain I–domain I interaction and serves as a hub for interaction with various proteins such as DnaB helicase and DiaA, which stimulates ATP–DnaA assembly at oriC ( 26 , 27 , 28 , 29 , 30 ). Two or three domain I molecules of the oriC– DnaA subcomplex bind a single DnaB hexamer, forming a stable higher-order complex ( 7 ). Domain II is a flexible linker ( 28 , 31 ).…”

“…Also, the Left- and Right-DnaA subcomplexes, which are oriented opposite to each other, could regulate bidirectional loading of DnaB helicases onto the ssDUE ( Fig. 1 B ) ( 7 , 8 , 35 ). Similarly, recent works suggest that the origin complex structure is bidirectionally organized in both archaea and eukaryotes ( 1 , 46 ).…”

Z-Ring-Associated Proteins Regulate Clustering of the Replication Terminus-Binding Protein ZapT in Caulobacter crescentus

“…Size exclusion chromatography. The size exclusion chromatography assay was performed essentially as described previously (57,62). Briefly, proteins were loaded onto a Superdex 200 PC3.2/30 column (2.4-ml column volume) equilibrated with SEC buffer (25 mM Tris-HCl [pH 7.5], 300 mM sodium chloride, and 20% sucrose) and fractionated at a flow rate of 20 ml/min, followed by SDS-15% PAGE and Coomassie brilliant blue staining.…”