title: Highly organized DnaA-oriC complexes recruit the single-stranded DNA for replication initiation.
abstract: In Escherichia coli, the replication origin oriC consists of two functional regions: the duplex unwinding element (DUE) and its flanking DnaA-assembly region (DAR). ATP-DnaA molecules multimerize on DAR, unwinding DUE for DnaB helicase loading. However, DUE-unwinding mechanisms and functional structures in DnaA-oriC complexes supporting those remain unclear. Here, using various in vitro reconstituted systems, we identify functionally distinct DnaA sub-complexes formed on DAR and reveal novel mechanisms in DUE unwinding. The DUE-flanking left-half DAR carrying high-affinity DnaA box R1 and the ATP-DnaA-preferential DnaA box R5, τ1-2 and I1-2 sites formed a DnaA sub-complex competent in DUE unwinding and ssDUE binding, thereby supporting basal DnaB loading activity. This sub-complex is further subdivided into two; the DUE-distal DnaA sub-complex formed on the ATP-DnaA-preferential sites binds ssDUE. Notably, the DUE-flanking, DnaA box R1-DnaA sub-complex recruits DUE to the DUE-distal DnaA sub-complex in concert with a DNA-bending nucleoid protein IHF, thereby promoting DUE unwinding and binding of ssDUE. The right-half DAR-DnaA sub-complex stimulated DnaB loading, consistent with in vivo analyses. Similar features are seen in DUE unwinding of the hyperthermophile, Thermotoga maritima, indicating evolutional conservation of those mechanisms.
authors: Department of Molecular Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
authors: Shogo
authors: S
authors: Ozaki
authors: Tsutomu
authors: T
authors: Katayama
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D001665:Binding Sites; D004261:DNA Replication; D004277:DNA, Single-Stranded; D004278:DNA, Superhelical / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D053488:DnaB Helicases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D034982:Integration Host Factors / Q000378:metabolism; D011485:Protein Binding; D018741:Replication Origin
chemical_list: D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; D004278:DNA, Superhelical; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria; D034982:Integration Host Factors; D000255:Adenosine Triphosphate; D053488:DnaB Helicases