Origin Remodeling and Opening in Bacteria Rely on Distinct Assembly States of the DnaA Initiator

Abstract

The initiation of DNA replication requires the melting of chromosomal origins to provide a template for replisomal polymerases. In bacteria, the DnaA initiator plays a key role in this process, forming a large nucleoprotein complex that opens DNA through a complex and poorly understood mechanism. Using structure-guided mutagenesis, biochemical, and genetic approaches, we establish an unexpected link between the duplex DNA-binding domain of DnaA and the ability of the protein to both self-assemble and engage singlestranded DNA in an ATP-dependent manner. Intersubunit cross-talk between this domain and the DnaA ATPase region regulates this link and is required for both origin unwinding in vitro and initiator function in vivo. These findings indicate that DnaA utilizes at least two different oligomeric conformations for engaging single-and double-stranded DNA, and that these states play distinct roles in controlling the progression of initiation.

Near-Atomic Structural Model for Bacterial DNA Replication Initiation Complex and its Functional Insights

“…Across the 20 total trajectories, however, we did not find any single structure that had maximal values of both N pp (= 4) and N pd (= 5) simultaneously. In these simulations, some DnaA molecules often dissociated from DNA, suggesting that DnaA domain III disturbs interactions between domain IV and DNA, which is consistent with the necessity of rigid body rotation of domain III upon binding to DNA (7,23,24).…”

“…Regarding the mechanisms of how DnaA facilitates unwinding of dsDNA, two models have been proposed. In one model, DnaA molecules, which do not take part in the replication initiation complex, bind to ssDUE and retain the unwound state (7,8). The other model is that DnaA molecules composing lefthalf subcomplex possess dual functions of dsDNA binding and ssDUE binding (9,10).…”

“…The resulting ssDNA is captured by DnaB helicase, followed by formation of the replisomes (2)(3)(4)(5). Molecular mechanisms of how DnaA facilitates dsDNA unwinding are still unclear, although some models have been proposed (1,(6)(7)(8)(9)(10). A high-resolution structure model of the initiation complex, discovered using computational modeling based on experimental data, would provide significant insight into the molecular mechanism.…”

“…Domain IV in these complexes, however, does not accommodate dsDNA. A rigid body rotation at the linker between domains III and IV has been suggested to enable this homooligomer to bind to dsDNA at domain IV (7,23,24). The N-terminal domain I contains a DnaB-binding site as well as a site for low-affinity domain I-domain I interaction (Fig.…”

Helicase Loading at Chromosomal Origins of Replication

“…These observations suggest that DnaA confines some of the single-stranded character of the plasmid to this AT-rich region of oriC. To provide a biochemical mechanism of unwinding, recent studies on the assembly of a DnaA oligomer as a right-handed filament suggest that positively charged and hydrophobic amino acids that line the interior of the DnaA filament interact with the negatively charged ssDNA Ozaki et al 2008;Duderstadt et al 2010Duderstadt et al , 2011Ozaki and Katayama 2011). Consistent with the ATP requirement for DnaAdependent origin unwinding, the formation of this DnaA filament requires the ATP-bound form of the enzyme.…”