Abstract
Chromosomal replication initiation requires dynamic mechanisms in higher-order nucleoprotein complexes that are constructed at the origin of replication. In Escherichia coli, DnaA molecules construct functional oligomers at the origin oriC, enabling localized unwinding of oriC and stable binding of DnaB helicases via multiple domain I molecules of oriC-bound DnaA. DnaA-bound DnaB helicases are then loaded onto the unwound region of oriC for construction of a pair of replisomes for bidirectional replication. However, mechanisms of DnaB loading to the unwound oriC remain largely elusive. In this study, we determined that His136 of DnaA domain III has an important role in loading of DnaB helicases onto the unwound oriC. DnaA H136A mutant protein was impaired in replication initiation in vivo, and in DnaB loading to the unwound oriC in vitro, whereas the protein fully sustained activities for oriC unwinding and DnaA domain I-dependent stable binding between DnaA and DnaB. Functional and structural analyses supported the idea that transient weak interactions between DnaB helicase and DnaA His136 within specific protomers of DnaA oligomers direct DnaB to a region in close proximity to single stranded DNA at unwound oriC bound to DnaA domain III of the DnaA oligomer. The aromatic moiety of His136 is basically conserved at corresponding residues of eubacterial DnaA orthologs, implying that the guidance function of DnaB is common to all eubacterial species.
Highlights
Chromosomal DNA replication is initiated by synergistic mechanisms involving multiple proteins with various functions
In E. coli, this step depends on dynamic interactions between the initiator DnaA and DnaB replicative helicase
In-depth biochemical characterization of DnaA H136A revealed that His136 is indispensable for DnaB loading at oriC, but that duplex unwinding element (DUE) unwinding, ssDUE binding, and assembly on oriC do not depend on this residue
Summary
Chromosomal DNA replication is initiated by synergistic mechanisms involving multiple proteins with various functions. The initial steps of replication in Escherichia coli occur at the unique replication origin, oriC, which has a sophisticated structure that directs unwinding of duplex DNA and loading of replicative helicases (Kaguni, 2011; Leonard and Grimwade, 2015; Wolanski et al, 2015; Katayama et al, 2017; Figure 1A) In these steps, the initiator DnaA molecules construct specific oligomers with the aid of DiaA (a DnaA-binding protein) and appropriately located DnaA-binding sequences (DnaA boxes) in the oriC DnaA-oligomerization region (DOR) (Leonard and Grimwade, 2015; Katayama et al, 2017). The unwound single-stranded (ss) DUE is stabilized by binding to the DnaA oligomer, which is a prerequisite for DnaB loading (Ozaki et al, 2008; Ozaki and Katayama, 2012; Sakiyama et al, 2017)
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