Schematic structures of oriC and DnaA and the replication initiation mechanism. (A) Overall structure of oriC with the AT-rich cluster. oriC (245 bp) includes the Duplex-Unwinding Element (DUE, purple bar) and the DnaA-Oligomerization Region (DOR, gray bar). DUE is composed of three AT-rich 13-mer repeats termed L, M, and R. Dark purple boxes indicate the specific sequences involved in DnaA-ssDNA binding (TT[A/G]T(T)). The AT-cluster (purple dots) flanking the DUE outside oriC is a supplementary unwinding region. The DOR contains 12 DnaA boxes (filled and open arrowheads representing sites with the full consensus TTA[A/T]NCACA sequence and sites with mismatches, respectively) and an IHF-binding region (IBR, green box). The DOR is subdivided into Left, Middle, and Right subregions. (B) Domains of DnaA. Domains I-IV are shown schematically, with amino acid residue numbers shown in each bracket. H/B motifs (V211 and R245, squares) and Arginine finger (R285, triangle) are indicated. The major functions of each domain are described on the right side of the structure. (C) DUE unwinding by the ssDUE recruitment mechanism. DnaA and IHF are indicated by red and green diagrams, respectively. When ATP-DnaA oligomerizes, DUE is unwound unstably by thermal motion and torsional stress. The M/R region of the DUE-upper strand (purple line) binds to R1-DnaA and R5M-DnaA via IHF-induced DNA bending, resulting in stable DUE unwinding. In addition to M/R-DUE, L-DUE is moderately unwound and interacts with the Right-DnaA subcomplex. (D) Mechanism for DnaB loading. Each Left-DnaA and Right-DnaA subcomplex binds to a DnaB hexamer (blue ovals) complexed with DnaC (yellow circles). (E) PriC-dependent fork rescue pathways. PriC (vivid red circle) promotes DnaB reloading to the abandoned fork, which exposes ssDNA regions. PriC binds to ssDNA at the replication fork, which remodels the SSB-ssDNA complex (for simplicity, SSB is omitted) for DnaC-dependent reloading of DnaB (upper panel). If the ssDNA region is accompanied with a nascent lagging strand, Rep helicase (green shape) is recruited by PriC and unwinds the lagging strand to expose the ssDNA region for DnaB loading (the lower case).

Requirement of priC for DNA replication initiation in dnaA46 and dnaC2 strains. (A) Cell growth abilities. MG1655 and its derivatives MIT125 (MG1655 dnaA46 tnaA::Tn10), KYA018 (MG1655 dnaC2 zjj18::cat), KRC002 (MG1655 ΔpriC::frt-kan), KRC004 (MG1655 dnaA46 tnaA::Tn10 ΔpriC::frt-kan), and KRC005 (MG1655 dnaC2 zjj18::cat ΔpriC::frt-kan) were grown overnight. 10-fold-serial dilutions of the overnight cultures (∼109 cells/mL) were incubated for 16 h at 30℃, or for 14 h at 35/37/42℃ on LB agar medium. Three independent experiments were performed. +, WT priC; -, ΔpriC::frt-kan. The images of representative plates are shown. (B) DNA replication initiation in synchronized dnaA46 cells with or without priC. Exponentially growing MIT125 (MG1655 dnaA46 tnaA::Tn10) (+) and KRC004 (MG1655 dnaA46 tnaA::Tn10 ΔpriC::frt-kan) (-) cells were synchronized to the pre-initiation stage by incubation for 90 min at 42℃ in LB medium, followed by incubation for 5 min at 30℃ to initiate DNA replication, which was further followed by incubation with rifampicin and cephalexin for 4 h at 42℃ to allow run-out of ongoing DNA replication. Samples were withdrawn after synchronization and run-out replication. The DNA contents of these samples were measured by flow cytometry. Representative histograms of three independent experiments are shown. For each sample, the mean number of cells with more than one chromosome equivalent after a 5 min release was quantified and is shown as a percentage with standard deviations. (C) DNA replication initiation in synchronized dnaC2 cells with or without priC. Exponentially growing KYA018 (MG1655 dnaC2 zjj18::cat) (+) and KRC005 (MG1655 dnaC2 zjj18::ca ΔpriC::frt-kan) (-) cells were synchronized to the pre-initiation stage by incubation for 80 min at 37℃ in LB medium, followed by incubation for 5 min at 30℃ and further incubation with rifampicin and cephalexin as described above. Samples were withdrawn and analyzed as described above.

Role of PriC in growth of DnaA mutant defective in interactions with DnaB. Cell growth of MIT125 (MG1655 dnaA46 tnaA::Tn10) and KRC004 (MG1655 dnaA46 tnaA::Tn10 ΔpriC::frt-kan) cells bearing pING1 plasmid vector (-) or its derivatives pKA234 expressing WT DnaA (WT) or pFH expressing DnaA F46A H136A (F46A H136A). Cells were grown at 30°C overnight and 10-fold-serial dilutions of the cultures (∼109 cells/mL) were incubated on LB agar plates containing ampicillin for 16 h at 30°C and for 14 h at 42°C, respectively. Four independent experiments were performed. +, WT priC; -, ΔpriC::frt-kan.

PriC stimulation of DNA replication initiation inhibited by DiaA oversupply. (A) Cell growth of MG1655, KRC002 (MG1655 ΔpriC::frt-kan), and FF001 (MG1655 Δrep::frt-kan) cells bearing pBR322 or pNA135 (pBR322 derivatives carrying diaA). Overnight cultures (∼109 cells/mL) of MG1655 and KRC002 cells bearing pBR322 or pNA135 were 10-fold serially diluted and incubated on LB agar plates containing ampicillin for 40 h at 25℃. The results of three independent experiments were consistent, and one which is shown. (B) Flow cytometry analyses of MG1655 and KRC002 (MG1655 ΔpriC::frt-kan) cells bearing pBR322 or pNA135. Cells were exponentially grown at 30℃ in LB medium with ampicillin, followed by further incubation with rifampicin and cephalexin to allow run-out of chromosomal DNA replication. DNA contents were quantified by flow cytometry, and cell size (mass) at the time of drug addition was measured by a Coulter counter. Mean mass, ori/mass ratio, doubling time (Td), and asynchrony index (A.I.: the percentage of cell numbers with non-2n copies of oriC per the cell numbers with 2n copies of oriC) of each strain are indicated at the top right of each panel with standard deviations. Three to four independent experiments were performed. +, WT priC; -, ΔpriC::frt-kan. (C) Flow cytometry analyses of MG1655, KRC003 (MG1655 ΔpriC::frt), SA103 (MG1655 ΔdiaA::frt-kan), and KRC006 (MG1655 ΔpriC::frt, ΔdiaA::frt-kan) cells. Cells were grown and analyzed as described above. Three independent experiments were performed. For “priC” column, +, WT priC; -, ΔpriC::frt. For “diaA” column, +, WT diaA; -, ΔdiaA::frt-kan.

Stimulation of replication initiation by PriC in Right-DnaA subcomplex-defective cells (A) Schematic structure of Left-oriC and R4Tma oriC. AT-rich region, DUE, and DOR are indicated by a gray dotted box, a gray box, and an open box, respectively. Filled and open arrowheads in the DOR show DnaA boxes with the full consensus sequence or mismatches. In addition, a striped box indicates the IBR. The regions of each mutant oriC are indicated below the structure as open boxes. The position of the TmaDnaA box substitution is indicated by a yellow arrowhead. (B) Cell growth of WT oriC and Left-oriC cells with or without priC. Overnight cultures (∼109 cells/mL) of NY20-frt (WT oriC; WT) and NY20L-frt (Left-oriC; Left) cells with (+) or without (-) priC were 10-fold serially diluted and incubated on LB agar plates for 24 h at 25°C, for 16 h at 30°C, and for 14 h at 37°C. +, WT priC; -, ΔpriC::frt-kan. Three independent experiments were performed. (C) Cell growth of WT oriC and oriC R4-box mutant cells with or without priC. NY20-frt (WT oriC) and NY24-frt (oriC with R4Tma substitution, R4Tma) cells with (+) or without (-) priC were grown as described in panel A. +, WT priC; -, ΔpriC::frt-kan. Four independent experiments were performed. (D) Flow cytometry analyses of WT and R4Tma cells with or without priC. Cells were exponentially grown at 30℃ in LB medium with ampicillin and analyzed as described in Figure 4. Representative histograms from four independent experiments are shown. Mean mass, ori/mass, and Td of each strain are indicated at the top right of each panel with standard deviations.

Stimulation of replication initiation by PriC in cells with a deletion in the ssDNA-expanding region of oriC (A) Schematic structure of subATL oriC as shown in Figure 5. The oriC region including the subATL oriC is indicated below the structure as a white box. (B) Cell growth of WT oriC and oriC subATL mutant cells with or without WT priC. NY20-frt (WT oriC) and NY20ATL-frt (oriC lacking AT and L sequences, subATL) with (+) or without (-) WT priC were grown as described in Figure 5. +, WT priC; -, ΔpriC::frt-kan. Three independent experiments were performed. (C) Flow cytometry analyses of WT oriC and oriC subATL mutant cells with or without WT priC. Cells were exponentially grown at 30℃ in LB medium and analyzed as described in Figure 4. Representative histograms from three independent experiments are shown. Mean mass, ori/mass, and Td of each strain are indicated at the top right of each panel with standard deviations.

DnaB loading onto the ssDUE by PriC. (A and B) In vitro reconstituted system for DnaB loading. Form I of oriC plasmid (pBSoriC; 1.6 nM) was incubated for 15 min at 30℃ with the indicated amount of PriC in the presence of ATP-DnaA or its mutant derivatives, DnaB, DnaC, IHF, gyrase, and SSB. The resulting plasmids were purified and analyzed using agarose gel electrophoresis. A representative image of three independent experiments is shown in the black/white-inverted mode (A). Band intensities of each lane in the gel image were analyzed by densitometric scanning. The percentages of form I* oriC plasmid per input DNA are shown as ‘Form I* (%)’. Mean and standard deviations are shown (B). Abbreviations in panel B: -, no addition of DnaA; WT, wild-type DnaA; FH, DnaA F46A H136A; VA, DnaA V211A. (C and D) In vitro reconstituted system containing DiaA. Form I of pBSoriC plasmid was used as described above except for the addition of the indicated amounts of DiaA. A representative image of three independent experiments is shown in black/white-inverted mode (C). Band intensities of each lane in the gel image were analyzed by densitometric scanning. The percentages of form I*oriC plasmid together with the mean and standard deviations are shown as described above (D).

PriC stimulation of cSDR-dependent cell growth. (A) Cell growth abilities. Cells of MIT125 (MG1655 dnaA46), MIT125c (MG1655 dnaA46 rnhA::cat), KRC004 (MG1655 dnaA46 ΔpriC::frt-kan), and KRC004c (MG1655 dnaA46 ΔpriC::frt-kan rnhA::cat) were grown at 30℃ overnight and 10-fold-serial dilutions of the overnight cultures (∼109 cells/mL) were incubated for 16 h at 30℃ and for 36 h at 40℃ on LB agar plates. Three independent experiments were performed. +, WT; -, deletion. (B) oriC and ter copy numbers. MIT125, MIT125c, KRC004, and KRC004c cells growing exponentially at 30℃ were further incubated for 90 min at 40℃ in LB medium. Samples were withdrawn before and after the 40℃ incubation. The genome DNA of each sample was extracted by boiling the cells for 5 min at 95℃. The relative copy numbers of oriC (84.6 min) and ter (32.4 min) to that of ypaB (50.5 min) were quantified using real-time qPCR. The data and averages of three independent experiments are shown with standard deviations.

Model for PriC-promoted replication initiation.

PriC-promoted rescue of blocked DnaA-oriC dependent replication initiation. oriC, DnaA, IHF, DnaB, and DnaC are illustrated as depicted in Figure 1C. When tight DnaA binding of DiaA (dark gray circle) (or DnaA F46A H136A double mutations) inhibits DnaB recruitment, PriC (Brilliant red circle) binds to a stably unwound DUE strand, recruiting a DnaB-DnaC complex to lower strand of DUE instead of Left-DnaA subcomplex. When a sufficient ssDNA region is exposed, PriC then recruits the DnaB-DnaC complex to the opposite strand.