A Common Mechanism for the ATP-DnaA-dependent Formation of Open Complexes at the Replication Origin

Abstract

Initiation of chromosomal replication and its cell cycle-coordinated regulation bear crucial and fundamental mechanisms in most cellular organisms. Escherichia coli DnaA protein forms a homomultimeric complex with the replication origin (oriC). ATPDnaA multimers unwind the duplex within the oriC unwinding element (DUE). In this study, structural analyses suggested that several residues exposed in the central pore of the putative structure of DnaA multimers could be important for unwinding. Using mutation analyses, we found that, of these candidate residues, DnaA Val-211 and Arg-245 are prerequisites for initiation in vivo and in vitro. Whereas DnaA V211A and R245A proteins retained normal affinities for ATP/ADP and DNA and activity for the ATPspecific conformational change of the initiation complex in vitro, oriC complexes of these mutant proteins were inactive in DUE unwinding and in binding to the single-stranded DUE. Unlike oriC complexes including ADP-DnaA or the mutant DnaA, ATPDnaA-oriC complexes specifically bound the upper strand of single-stranded DUE. Specific T-rich sequences within the strand were required for binding. The corresponding conserved residues of the DnaA ortholog in Thermotoga maritima, an ancient eubacterium, were also required for DUE unwinding, consistent with the idea that the mechanism and regulation for DUE unwinding can be evolutionarily conserved. These findings provide novel insights into mechanisms for pore-mediated origin unwinding, ATP/ADP-dependent regulation, and helicase loading of the initiation complex.Initiation of chromosomal replication and its cell cycle-coordinated regulation bear crucial and fundamental mechanisms in most cellular organisms. In Escherichia coli, DnaA forms a stable complex with ATP or ADP and binds to 9-mer sequences called DnaA boxes within the replication origin oriC, resulting in the formation of homomultimeric complexes (1-4). A DnaA-binding protein, DiaA, directly stimulates formation of ATP-DnaA multimers on oriC (5, 6). ATP-DnaA multimers, but not ADP-DnaA multimers, promote specific inter-DnaA interactions on oriC, resulting in the adoption of an activated conformation as the initiation complexes, which interact with ATP-DnaA-specific low affinity sites within oriC (7-9). This conformational change triggers duplex unwinding of the ATrich 13-mer repeats (DNA unwinding element (DUE) 4 ) within oriC with the aid of the superhelicity of DNA and heat energy, creating open complexes (10, 11). The mechanisms and functional structures within DnaA directly responsible for the ATPDnaA-specific duplex unwinding remain unexplored.Open complex formation is a critical regulatory point for determining whether replicational initiation will occur during the cell cycle (1, 2). DnaB helicase is loaded onto the singlestranded (ss) region in open complexes in a manner depending on a DnaA-DnaB interaction and the DnaC helicase loader. The loaded helicase expands the ssDNA region, which leads to the assembly of replication machineries, including DnaG primase and ...

The structure of a DnaA/HobA complex from Helicobacter pylori provides insight into regulation of DNA replication in bacteria

“…Its modular architecture allows for interactions with different binding partners during the various steps of the formation of the active replication forks and coupling to other cellular events (3,15,17,20,22,29,30). In E. coli, DnaA III interactions with itself, Hda, or DnaC are important for the spatial positioning of the ATP-DnaA molecules and their recycling (13,17,31,32). Protein-protein interactions involving DnaA I-II play critical roles in DnaA self-oligomerization, helicase loading (7,10,17), and regulation of the replication timing (16,28).…”

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

“…In our simulation model of the left-half subcomplex, the modeled DNA terminus proximal to DUE orients toward the pentameric DnaA AAA+ helix, despite the absence of an explicit DUE region. Additionally there seems to be enough space for ssDUE entering around DnaA V211 and R245, which are known to be important residues for ssDUE binding (6). Thus, the current model suggests that the second model is possible in E. coli.…”

“…1B) (14,24). The helical oligomer of a AAA+ domain can bind ssDNA by specific residues exposed on its central path (6,8). Crystal structures have been solved for helically arranged homooligomer of domains III and IV of Aquifex aeolicus DnaA with and without bound ssDNA (8,24).…”

“…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.…”

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.…”