Background: Multiple DnaA molecules form highly ordered complexes on the origin DNA to initiate chromosomal replication. Results: Novel structural motifs of DnaA are specifically required for the formation of the DNA unwinding-specific DnaA subcomplex. Conclusion: Distinct inter-DnaA interactions are required for the unwinding-specific subcomplex. Significance: Differentiation of the unwinding-specific subcomplex and a key mechanism underlying it are revealed.

SignificanceDuring initiation of DNA replication in eukaryotes, the origin recognition complex, with Cdc6 and Cdt1, assembles an inactive Mcm2-7 double hexamer on the dsDNA. Later, the double hexamer recruits Cdc45 and GINS to form two active and separate DNA helicases. The active Cdc45–Mcm2-7–GINS helicase encircles the leading strand while excluding the lagging strand. One of the fundamental unanswered questions is how each Mcm2-7 hexamer converts from binding dsDNA to binding one of the single strands. The structure of the double hexamer on dsDNA reveals how DNA interacts with key elements inside the central channel, leading us to propose a lagging-strand extrusion mechanism. This work advances our understanding of eukaryotic replication initiation.

Background: ATP-DnaA molecules oligomerize and form two subcomplexes on the replication origin. Results: The Arg fingers of DnaA bound at the outer edges of the DnaA complexes are oriented inward within the origin. Conclusion: The Arg fingers, but not bound ATP, of the outer edge DnaA protomers promote construction of active initiation complexes. Significance: An important mechanical basis in the initiation complex is revealed.

In Escherichia coli, the level of the ATP–DnaA initiator is increased temporarily at the time of replication initiation. The replication origin, oriC, contains a duplex-unwinding element (DUE) flanking a DnaA-oligomerization region (DOR), which includes twelve DnaA-binding sites (DnaA boxes) and the DNA-bending protein IHF-binding site (IBS). Although complexes of IHF and ATP–DnaA assembly on the DOR unwind the DUE, the configuration of the crucial nucleoprotein complexes remains elusive. To resolve this, we analyzed individual DnaA protomers in the complex and here demonstrate that the DUE–DnaA-box-R1–IBS–DnaA-box-R5M region is essential for DUE unwinding. R5M-bound ATP–DnaA predominantly promotes ATP–DnaA assembly on the DUE-proximal DOR, and R1-bound DnaA has a supporting role. This mechanism might support timely assembly of ATP–DnaA on oriC. DnaA protomers bound to R1 and R5M directly bind to the unwound DUE strand, which is crucial in replication initiation. Data from in vivo experiments support these results. We propose that the DnaA assembly on the IHF-bent DOR directly binds to the unwound DUE strand, and timely formation of this ternary complex regulates replication initiation. Structural features of oriC support the idea that these mechanisms for DUE unwinding are fundamentally conserved in various bacterial species including pathogens.

Upon DNA replication initiation in Escherichia coli, the initiator protein DnaA forms higher-order complexes with the chromosomal origin oriC and a DNA-bending protein IHF. Although tertiary structures of DnaA and IHF have previously been elucidated, dynamic structures of oriC-DnaA-IHF complexes remain unknown. Here, combining computer simulations with biochemical assays, we obtained models at almost-atomic resolution for the central part of the oriC-DnaA-IHF complex. This complex can be divided into three subcomplexes; the left and right subcomplexes include pentameric DnaA bound in a head-to-tail manner and the middle subcomplex contains only a single DnaA. In the left and right subcomplexes, DnaA ATPases associated with various cellular activities (AAA+) domain III formed helices with specific structural differences in interdomain orientations, provoking a bend in the bound DNA. In the left subcomplex a continuous DnaA chain exists, including insertion of IHF into the DNA looping, consistent with the DNA unwinding function of the complex. The intervening spaces in those subcomplexes are crucial for DNA unwinding and loading of DnaB helicases. Taken together, this model provides a reasonable near-atomic level structural solution of the initiation complex, including the dynamic conformations and spatial arrangements of DnaA subcomplexes.DnaA | molecular simulation | coarse-grained model | oriC C hromosomal DNA replication is initiated by unwinding the dsDNA of the replication origin, which requires formation of higher-order protein-DNA complexes, typically referred to as the initiation complexes (1). DnaA is a major replication initiation protein conserved in the initiation complex of most eubacterial species. In a model organism, Escherichia coli, DnaA forms homooligomers on the replication origin oriC, which promotes dsDNA unwinding. The resulting ssDNA is captured by DnaB helicase, followed by formation of the replisomes (2-5). Molecular mechanisms of how DnaA facilitates dsDNA unwinding are still unclear, although some models have been proposed (1, 6-10). 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.The E. coli minimal oriC region contains the AT-rich DNA unwinding element (DUE), at least 11 DnaA-binding motifs (termed DnaA boxes) and a single binding site for the integration host factor (IHF) (Fig. 1A) (1-5, 11-15). The DnaA boxes contain 9-mer nucleotides (consensus sequence TTATNCACA, where N can be any base) (16). The 11 DnaA boxes (R1-2, R4, R5M, I1-3, C1-3, and τ2) have differing affinities to DnaA and motif orientations (indicated by triangles in Fig. 1A): The two terminal boxes R1 and R4 have especially high affinities (dissociation constants 1-6 nM for R1 and ∼1 nM for R4), whereas others have modest (R2) to low affinities (I1-3, C1-3, and R5M and τ2; dissociation constants for R5M are >200 nM) (12-19). The 11 DnaA boxes have been divided into two groups...

DNA replication results in the doubling of the genome prior to cell division. This process requires the assembly of 50 or more protein factors into a replication fork. Here, we review recent structural and biochemical insights that start to explain how specific proteins recognize DNA replication origins, load the replicative helicase on DNA, unwind DNA, synthesize new DNA strands, and reassemble chromatin. We focus on the minichromosome maintenance (MCM2-7) proteins, which form the core of the eukaryotic replication fork, as this complex undergoes major structural rearrangements in order to engage with DNA, regulate its DNA-unwinding activity, and maintain genome stability.

In Escherichia coli, bidirectional chromosomal replication is accompanied by the colocalization of sister replication forks. However, the biological significance of this mechanism and the key factors involved are still largely unknown. In this study, we found that a protein, termed CrfC, helps sustain the colocalization of nascent DNA regions of sister replisomes and promote chromosome equipartitioning. CrfC formed homomultimers that bound to multiple molecules of the clamp, a replisome subunit that encircles DNA, and colocalized with nascent DNA regions in a clamp-binding-dependent manner in living cells. CrfC is a dynamin homolog; however, it lacks the typical membrane-binding moiety and instead possesses a clamp-binding motif. Given that clamps remain bound to DNA after Okazaki fragment synthesis, we suggest that CrfC sustains the colocalization of sister replication forks in a unique manner by linking together the clamp-loaded nascent DNA strands, thereby laying the basis for subsequent chromosome equipartitioning.

The initiation of bacterial chromosomal replication is regulated by multiple pathways. To explore novel regulators, we isolated multicopy suppressors for the cold-sensitive hda-185 ΔsfiA(sulA) mutant. Hda is crucial for the negative regulation of the initiator DnaA and the hda-185 mutation causes severe replication overinitiation at the replication origin oriC. The SOS-associated division inhibitor SfiA inhibits FtsZ ring formation, an essential step for cell division regulation during the SOS response, and ΔsfiA enhances the cold sensitivity of hda-185 cells in colony formation. One of the suppressors comprised the yfdQ-yfdR-yfdS-yfdT gene cluster carried on a cryptic prophage. Increased copy numbers of yfdQRT or yfdQRS inhibited not only hda-185-dependent overinitiation, but also replication overinitiation in a hyperactive dnaA mutant, and in a mutant lacking an oriC-binding initiation-inhibitor SeqA. In addition, increasing the copy number of the gene set inhibited the growth of cells bearing specific, initiation-impairing dnaA mutations. In wild-type cells, multicopy supply of yfdQRT or yfdQRS also inhibited replication initiation and increased hydroxyurea (HU)-resistance, as seen in cells lacking DiaA, a stimulator of DnaA assembly on oriC. Deletion of the yfdQ-yfdR-yfdS-yfdT genes did not affect either HU resistance or initiation regulation. Furthermore, we found that DnaA bound specifically to YfdR in soluble protein extracts oversupplied with YfdQRST. Purified YfdR also bound to DnaA, and DnaA Phe46, an amino acid residue crucial for DnaA interactions with DiaA and DnaB replicative helicase was important for this interaction. Consistently, YfdR moderately inhibited DiaA-DnaA and DnaB-DnaA interactions. In addition, protein extracts oversupplied with YfdQRST inhibited replication initiation in vitro. Given the roles of yfdQ and yfdS in cell tolerance to specific environmental stresses, the yfdQ-yfdR-yfdS-yfdT genes might downregulate the initiator DnaA-oriC complex under specific growth conditions.