Tsutomu Katayama

The Escherichia coli chromosomal origin, oriC, contains a duplex-unwinding element (DUE) flanked by two clusters of the initiator DnaA-binding sites (DnaA boxes). ATP-DnaA oligomerizes to construct Left- and Right–DnaA subcomplexes on these clusters. These complexes coordinately promote DUE unwinding and DnaB helicase loading onto the unwound region for replication initiation. However, despite the strong conservation of DnaA and DnaB across bacterial species, oriC architectures vary largely. Remarkably, bipartite and bilateral configurations contain the two DnaA box-clusters separated by an insertion of the dnaA gene or positioned on both sides of the DUE, respectively. Here, we demonstrate that E. coli oriC variants mimicking these configurations remain fundamentally functional. Notably, DnaB loading activity of these variants showed increased dependence on single-stranded DUE binding by the relocated DnaA subcomplex and on the AT cluster upstream of the DUE, reflecting reduced stability of DUE unwinding. Consistent with these in vitro results, these variants supported cellular replication initiation with only slow cell growth rates. Together, these findings reveal remarkable architectural flexibility, mechanistic robustness, and a conserved mechanism in oriC. Also, those highlight unwinding stability as a key determinant of oriC adaptation to rapid cell growth, providing insight into the evolution of oriC architecture.

In model bacteria, initiation of chromosome replication requires engagement of single-stranded DNA by oligomers of the DnaA-family initiator assembled within the origin DNA. Although arrays of double-strand motifs recognized by DnaA are a general feature of the origins, the DnaA-binding single-strand elements are elucidated in only a limited number of species, and the mechanical principles governing their recognition remain elusive. Using the Alphaproteobacterium Caulobacter crescentus , we identify a previously uncharacterized GA-rich single-stranded element in the origin that directly engages DnaA oligomers and is essential for robust initiation. This element is positioned at a subkilobase distance from the DnaA oligomerization region and is brought into proximity through dynamic structural rearrangements. Moreover, DnaA oligomers exhibit an unexpectedly broad yet constrained capacity to accommodate single-stranded sequence variation. These findings provide the molecular basis for origin plasticity, highlighting how origins can diverge while preserving initiation logic.

Bidirectional replication is a conserved principle requiring coordinated translocation of the two replicative helicases loaded at the origin. In Escherichia coli, the initiator protein DnaA constructs two oligomers in an initiation complex at the origin oriC, which locally unwinds the DNA and recruits DnaB helicase-DnaC loader complexes to the unwound region. We previously demonstrated that the two DnaA subcomplexes constructed on oriC bind a specific DNA strand of the unwound origin and tether individual DnaB–DnaC complexes via stable interactions between DnaA domain I and DnaB. A low-affinity DnaA–DnaB interaction mediated by DnaA domain III His136 is essential for DnaB-dependent origin unwinding. Here, we identified DnaB Thr86 as the critical residue mediating this low-affinity interaction. Structural modeling suggests that Thr86 is surface-exposed near the DNA entry site of DnaB. Functional analyses revealed that DnaB Thr86 was specifically required for DnaB loading onto the DnaA-bound strand of the unwound oriC. Furthermore, this strand-specific DnaB loading was required for enabling translocation of the opposing DnaB helicase loaded on the DnaA-free strand. Our findings define a novel mechanism of strand-specific helicase loading, mediated by the low-affinity DnaA–DnaB interactions, which promotes the coordinated translocation of the loaded DnaB helicases, ensuring bidirectional replication from oriC.

Bidirectional replication is a conserved principle requiring the coordinated loading and activation of two replicative helicases at the origin. In Escherichia coli , the initiator protein DnaA constructs a higher-order initiation complex at the origin oriC which locally unwinds the DNA, and recruits DnaB helicase-DnaC loader complexes to the unwound region. We previously demonstrated that the two DnaA subcomplexes formed on oriC bind a specific DNA strand of the unwound origin, and tether individual DnaB-DnaC complexes via stable interactions between DnaA domain I and DnaB. A low-affinity DnaA-DnaB interaction mediated by DnaA domain III His136 is essential for DnaB-dependent origin unwinding. Here, we identified DnaB Thr86 as the critical residue mediating this low-affinity interaction. Structural modelling suggests that Thr86 is surface-exposed near the DNA entry site of DnaB. Functional analyses revealed that DnaB Thr86 was specifically required for DnaB loading onto the DnaA-bound strand of the unwound oriC . Furthermore, this strand-specific DnaB loading was required for enabling translocation of the opposing DnaB helicase loaded on the DnaA-free strand. Our findings define a novel regulatory mechanism of strand-specific helicase loading, mediated by the low-affinity DnaA–DnaB interactions, which ensures the coordinated loading and translocation of DnaB helicases for bidirectional replication from oriC .

In Escherichia coli, replisome and replication fork assembly is initiated by DnaB helicase loading at the chromosomal origin oriC via its interactions with the DnaA initiator and the DnaC helicase loader. Upon replication fork arrest, the replisome including DnaB dissociates from the stalled fork. Replication fork progression is rescued by primosomal protein PriA- or PriC-dependent pathway in which PriA and PriC promote reloading of DnaB in different mechanisms. However, the mechanism responsible for rescue of blocked replication initiation at oriC remains unclear. Here, we found that PriC rescued blocked replication initiation in cells expressing an initiation-specific DnaC mutant, in mutant cells defective in DnaA-DnaB interactions, and in cells containing truncated oriC sequence variants. PriC rescued DnaB loading at oriC even in the absence of Rep helicase, a stimulator of the PriC-dependent replication fork restart pathway. These results of in vitro reconstituted assays concordantly suggest that this initiation-specific rescue mechanism provides a bypass of the DnaA-DnaB interaction for DnaB loading by PriC-promoted loading of DnaB to the unwound oriC region. These findings expand understanding of mechanisms sustaining the robustness of replication initiation and specific roles for PriC in the genome maintenance.

In Escherichia coli, replisome and replication fork assembly is initiated by DnaB helicase loading at the chromosomal origin oriC via its interactions with the DnaA initiator and the DnaC helicase loader. Upon replication fork arrest, the replisome including DnaB dissociates from the stalled fork. Replication fork progression is rescued by primosomal protein PriA- or PriC-dependent pathway in which PriA and PriC promote reloading of DnaB in different mechanisms. However, the mechanism responsible for rescue of blocked replication initiation at oriC remains unclear. Here, we found that PriC rescued blocked replication initiation in cells expressing an initiation-specific DnaC mutant, in mutant cells defective in DnaA-DnaB interactions, and in cells containing truncated oriC sequence variants. PriC rescued DnaB loading at oriC even in the absence of Rep helicase, a stimulator of the PriC-dependent replication fork restart pathway. These and results of in vitro reconstituted assays concordantly suggest that this initiation-specific rescue mechanism provides a bypass of the DnaA-DnaB interaction for DnaB loading by PriC-promoted loading of DnaB to the unwound oriC region. These findings expand understanding of mechanisms sustaining the robustness of replication initiation and specific roles for PriC in the genome maintenance.

The 12th 3R + 3C international symposium focused on cutting‐edge research into the molecular mechanisms and regulatory systems of DNA replication, repair, and recombination (3R) as well as those of chromatin dynamics, chromosome architecture, and the cell cycle (3C). It also covered pioneering research into how these processes control cell growth, cell homeostasis, differentiation, development, and aging, in addition to how they contribute to diseases such as cancer, chromosomal abnormalities, and evolution of organisms. In terms of methodology, the symposium highlighted new trends in single‐molecule/single‐cell analysis, cryo‐electron microscopy analysis, kinetic analysis of higher‐order protein complexes, informatic analysis of genome dynamics, and new mathematical and theoretical analyses. Held in Fukuoka City center from November 18 to 22, 2024, this symposium attracted about 250 participants, including approximately 150 from Japan and nearly 100 from overseas. To foster mutual understanding and exchange between different fields, all the oral presentations took place in a single conference hall throughout the symposium. This format facilitated active and in‐depth discussions among participants, including young researchers, graduate students, and postdoctoral fellows.

Oscillation of the active form of the initiator protein DnaA (ATP-DnaA) allows for the timely regulation for chromosome replication. After initiation, DnaA-bound ATP is hydrolyzed, producing inactive ADP-DnaA. For the next round of initiation, ADP-DnaA interacts with the chromosomal locus DARS2 bearing binding sites for DnaA, a DNA-bending protein IHF, and a transcription activator Fis. The IHF binding site is about equidistant between the DnaA and Fis binding sites within DARS2. The DARS2-IHF-Fis complex promotes ADP dissociation from DnaA and furnishes ATP-DnaA at the pre-initiation stage, which dissociates Fis in a negative-feedback manner. However, regulation for IHF binding as well as mechanistic roles of Fis and specific DNA structure at DARS2 remain largely unknown. We have discovered that negative DNA supercoiling of DARS2 is required for stimulating IHF binding and ADP dissociation from DnaA in vitro. Consistent with these, novobiocin, a DNA gyrase inhibitor, inhibits DARS2 function in vivo. Fis Gln68, an RNA polymerase-interaction site, is suggested to be required for interaction with DnaA and full DARS2 activation. Based on these and other results, we propose that DNA supercoiling activates DARS2 function by stimulating stable IHF binding and DNA loop formation, thereby directing specific Fis–DnaA interaction.

For bidirectional replication in Escherichia coli, higher order complexes are formed at the replication origin oriC by the initiator protein DnaA, which locally unwinds the left edge of oriC to promote the loading of two molecules of DnaB helicases onto the unwound region via dynamic interactions with the helicase-loader DnaC and the oriC-bound DnaA complex. One of the two helicases must translocate rightwards through oriC-bound DnaA complex. Here, we used a synthetic forked oriC DNA, which mimics the unwound state of oriC, to examine DnaB translocation through the oriC-bound DnaA complex. We found that DnaB helicase alone cannot pass through the oriC-bound DnaA complex without the help of single-strand binding protein (SSB). In the presence of SSB, DnaB passed through this complex along with its helicase function, releasing DnaA molecules. In addition, DnaB helicase activity is known to be inhibited by oversupply of DnaC, but this inhibition was relieved by SSB. These results suggest a mechanism that when two DnaB helicases are loaded at oriC, one translocates leftwards to expand the DnaA-unwound region and allows SSB binding to the single-stranded DNA, and such SSB molecules then stimulate translocation of the other helicase rightwards through the oriC-bound DnaA complex.

In Escherichia coli , replisome and replication fork assembly is initiated by DnaB helicase loading at the chromosomal origin oriC via its interactions with the DnaA initiator and the DnaC helicase loader. Upon replication fork arrest, the replisome including DnaB dissociates from the stalled fork. Replication fork progression is rescued by primosomal protein PriA- or PriC-dependent pathway in which PriA and PriC promote reloading of DnaB in different mechanisms. However, the mechanism responsible for rescue of blocked replication initiation at oriC remains unclear. Here, we found that PriC rescued blocked replication initiation in cells expressing an initiation-specific DnaC mutant, in mutant cells defective in DnaA-DnaB interactions, and in cells containing truncated oriC sequence variants. PriC rescued DnaB loading at oriC even in the absence of Rep helicase, a stimulator of the PriC-dependent replication fork restart pathway. These and results of in vitro reconstituted assays concordantly suggest that this initiation-specific rescue mechanism provides a bypass of the DnaA-DnaB interaction for DnaB loading by PriC-promoted loading of DnaB to the unwound oriC region. These findings expand understanding of mechanisms sustaining the robustness of replication initiation and specific roles for PriC in the genome maintenance.