Interdependent progression of bidirectional sister replisomes in E. coli

  1. Po Jui Chen
  2. Anna B McMullin
  3. Bryan J Visser
  4. Qian Mei
  5. Susan M Rosenberg
  6. David Bates  Is a corresponding author
  1. Molecular Virology and Microbiology, Baylor College of Medicine, United States
  2. Graduate Program in Integrative Molecular and Biomedical Sciences, Baylor College of Medicine, United States
  3. Systems, Synthetic, and Physical Biology Program, Rice University, United States
  4. Molecular and Human Genetics, Baylor College of Medicine, United States
  5. Dan L Duncan Comprehensive Cancer Center, Baylor College of Medicine, United States
6 figures, 1 table and 1 additional file

Figures

Replisome splitting.

Under slow growth conditions, E. coli bidirectional sister replisomes (yellow spheres) are initially colocalized, then separate part way through the replication period, marking a transition from …

Figure 2 with 2 supplements
Sister replisome association under slow and fast growth conditions.

(A) Three-dimensional (3D) replisome imaging. E. coli cells with an SSB-YPet tag were grown to mid-exponential phase in M9 minimal media at 37°C then imaged at 0.2 µm z-plane increments, …

Figure 2—figure supplement 1
Timeline and spatial dynamics of replication under the Splitting with cousins model.

Arrangement of replisomes and DNA in cell drawings are speculative and are based solely on measurements of genomic content and number of DnaN-YPet foci per cell (Figure 2H, I).

Figure 2—figure supplement 2
Replisome dynamics under fast growth conditions using a monomeric DnaN-mCherry fusion protein.

(A) Image of DnaN-mCherry-expressing wild-type cells (DB2962) under fast growth conditions. (B) Distribution of DnaN-mCherry foci per cell. (C) Fitness of replisome modeling as in Figure 2K. (D) …

Figure 3 with 4 supplements
Measuring the effect of replisome pairing on fork progression by whole-genome sequencing.

(A) Experimental approach. Strains carrying an inducible tetO array replication barrier (top) are roadblocked and progression of the unblocked orphaned replisome is quantified by sequencing. …

Figure 3—figure supplement 1
Individual sequencing profiles under slow growth conditions.

Profiles along the blocked chromosome arm are approximately flat between the tetO array and the most distal ter site, which terminates replication emanating from the unblocked arm. Profile noise is …

Figure 3—figure supplement 2
Individual sequencing profiles under fast growth conditions.

Slight slope on the blocked chromosome arm downstream of the tetO array indicates partial ‘read-through’ of opposite arm forks through the terminus region, eventually stopping at the tetO array. …

Figure 3—figure supplement 3
Time course of roadblocking under fast growth conditions.

(A) Sequencing profiles for +0.13 Mb roadblock (DB2185) every 30 min after TetR-YFP induction (n = 1). (B) Change in progression of the unblocked (left) replisome after roadblocking relative to time …

Figure 3—figure supplement 4
Imaging replisomes in roadblocked cells under fast growth conditions.

DB2972 cells were roadblocked at +0.13 Mb from oriC for 2 hr and imaged for replisomes (red, DnaN-mCherry) and the roadblock (yellow, TetR-YFP). Multiple dispersed replisome foci rarely colocalizing …

Figure 4 with 2 supplements
The effect of replisome pairing on replication fork velocity.

(A) Replication elongation measured as a change in cellular DNA content over time after inhibiting replication initiation with rifampicin (RIF) and cell division with cephalexin (CEP). (Top) …

Figure 4—figure supplement 1
DNA histograms for all samples.

DNA histograms are shown at 5-min intervals with 90-min time point overlayed (gray). Strains are MG1655 (No block), DB2146 (+1.10 Mb block), DB3195 (+0.63 Mb block), DB3216 (−0.35 Mb block), and …

Figure 4—figure supplement 2
Cumulative curve plots for all samples.

Cumulative curves of DNA histograms (Figure 4—figure supplement 1) with 90-min time point overlayed (gray).

Figure 5 with 1 supplement
The requirement of RecB-dependent fork restart in uncoupled replisomes.

(A) Raw sequencing profiles of the unblocked chromosome arm in recB+ and recB cells after rifampicin runout. Strains contained either no block (wild-type MG1655 or its recB derivative), an oriC-dis…

Figure 5—figure supplement 1
Individual sequencing profiles after rifampicin runout.

Data as described in Figure 5A. Strains are MG1655 (recB+, No block), DB2257 (recB, No block), DB2185 (recB+, +0.13 Mb block), DB2403 (recB, +0.13 Mb block), DB2193 (recB+, +0.34 Mb block), DB2722 …

Figure 6 with 3 supplements
Holiday junction frequency on DNA replicated by uncoupled replisomes.

(A) Genome binding of the Holliday junction protein RuvCDef-GFP (RDG) by chromatin immunoprecipitation and sequencing (ChIP-seq). RDG-expressing cells either contained no roadblock (DB2945) or were …

Figure 6—figure supplement 1
Distribution of RuvCDef-GFP (RDG) binding.

Frequency indicates the percentage of RDG chromatin immunoprecipitation and sequencing (ChIP-seq) IP/input values in 0.2-increment bins for wild-type DB2945 (No RB), DB3124 (+1.10 Mb blocked), …

Figure 6—figure supplement 2
RuvCDef-GFP (RDG)-binding peaks in oriC-proximal roadblocked cells.

Unblocked chromosome arm is shown between −2 Mb and oriC. Peaks (dashed lines) are locations of RDG signal ≥4 standard deviation (s.d.) above the 100 kb local median.

Figure 6—figure supplement 3
Genomic binding of RuvCDef-GFP (RDG) and major nucleoid proteins.

(A) Nucleoid protein-binding profiles (black) and consensus RDG-binding peaks (dashed lines). (B) Correlation plots of nucleoid protein and RDG binding (average +0.13 Mb, +0.34 Mb blocked cells). …

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Strain, strain background (Escherichia coli)AB1157 dnaN-mCherry FRTKanFRTMoolman et al., 2014BN1682
Strain, strain background (E. coli)AB1157 dnaN-ypet FRTThis paperDB1568RRL190 flipped to KmS w/pCP20
Strain, strain background (E. coli)MG1655 141x tetO array @ lac, pDM15 (+1.10 Mb block)Joshi et al., 2011DB2146
Strain, strain background (E. coli)MG1655 141x tetO array @ glnA, pDM15 (+0.13 Mb block)Joshi et al., 2011DB2185
Strain, strain background (E. coli)MG1655 141x tetO array @ dnaB, pDM15 (+0.34 Mb block)Joshi et al., 2011DB2193
Strain, strain background (E. coli)MG1655 ΔrecB FRTKanFRTThis paperDB2257MG1655 × P1.JW2788
Strain, strain background (E. coli)MG1655 141x tetO array @ gln, ΔrecB FRTKanFRT, pDM15 (+0.13 Mb block)This paperDB2403DB2185 × P1.JW2788
Strain, strain background (E. coli)MG1655 141x tetO array @ dnaB, ΔrecB FRTKanFRT, pDM15 (+0.34 Mb block)This paperDB2722DB2193 × P1.JW2788
Strain, strain background (E. coli)MG1655 141x tetO array @ lac, ΔrecB FRTKanFRT, pDM15 (+1.10 Mb block)This paperDB2725DB2146 × P1.JW2788
Strain, strain background (E. coli)MG1655 dnaN-mCherry FRTKanFRTThis paperDB2943MG1655 × P1.BN1682
Strain, strain background (E. coli)MG1655 λcIts857 PR::ruvCDef-gfp FRTKanFRT (42°C inducible RDG cassette)This paperDB2945SMR19379 recombineered with primers P1/P2; P3/P4
Strain, strain background (E. coli)MG1655 141x tetO array @ gln, λcIts857 PR::ruvCDef-gfp FRTKanFRT, pDM15 (+0.13 Mb block, RDG)This paperDB2954DB2185 × P1.DB2945
Strain, strain background (E. coli)MG1655 141x tetO array @ dnaB, λcIts857 PR::ruvCDef-gfp FRTKanFRT, pDM15 (+0.34 Mb block, RDG)This paperDB2956DB2193 × P1.DB2945
Strain, strain background (E. coli)MG1655 dnaN-mCherry FRTThis paperDB2962DB2943 flipped to KmS w/pCP20
Strain, strain background (E. coli)MG1655 141x tetO array @ glnA, pDM15 (+0.13 Mb block) dnaN-mCherry FRTKanFRTThis paperDB2970DB2185 × P1.BN1682
Strain, strain background (E. coli)MG1655 141x tetO array @ glnA, pDM15 (+0.13 Mb block) dnaN-mCherry FRTThis paperDB2972DB2970 flipped to KmS w/pCP20
Strain, strain background (E. coli)BW25113 yjiC::141x tetO array FRTThis paperDB3094JW4288 × pJZ087 & pCP20
Strain, strain background (E. coli)BW25113 yjbL::141x tetO array FRTThis paperDB3096JW4007 × pJZ087 & pCP20
Strain, strain background (E. coli)MG1655 141x tetO array @ lac, λcIts857 PR::ruvCDef-gfp FRTKanFRT, pDM15 (+1.10 Mb block, RDG)This paperDB3124DB2146 × P1.DB2945
Strain, strain background (E. coli)MG1655 yjiC::141x tetO array FRTThis paperDB3191MG16556 × P1.DB3094
Strain, strain background (E. coli)MG1655 yjbL::141x tetO array FRTThis paperDB3193MG1655 × P1.DB3096
Strain, strain background (E. coli)MG1655 yjiC::141x tetO array FRT, pDM15 (+0.63 Mb block)This paperDB3195DB3191 × pDM15
Strain, strain background (E. coli)MG1655 yjbL::141x tetO array FRT pDM15 (+0.32 Mb block)This paperDB3198DB3193 × pDM15
Strain, strain background (E. coli)BW25113 yhgN::141x tetO array FRTThis paperDB3212JW3397 × pJZ087 & pCP20
Strain, strain background (E. coli)MG1655 yhgN::141x tetO array FRTThis paperDB3214MG1655 × P1.3212
Strain, strain background (E. coli)MG1655 yhgN::141x tetO array FRT, pDM15 (−0.35 Mb block)This paperDB3216DB3214 × pDM15
Strain, strain background (E. coli)BW25113 yjgZ::141x tetO array FRTThis paperDB3258JW4236 × pJZ087 & pCP20
Strain, strain background (E. coli)MG1655 yjgZ::141x tetO array FRTThis paperDB3262MG1655 × P1.DB3258
Strain, strain background (E. coli)MG1655 yjgZ::141x tetO array FRT, pDM15 (+0.58 Mb block)This paperDB3264DB3262 × pDM15
Strain, strain background (E. coli)BW25113 ΔrecB FRTKanFRTBaba et al., 2006JW2788Keio collection
Strain, strain background (E. coli)BW25113 ∆yhgN FRTKanFRTBaba et al., 2006JW3397Keio collection
Strain, strain background (E. coli)BW25113 ∆yjbL FRTKanFRTBaba et al., 2006JW4007Keio collection
Strain, strain background (E. coli)BW25113 ∆yjgZ FRTKanFRTBaba et al., 2006JW4236Keio collection
Strain, strain background (E. coli)BW25113 ∆yjiC FRTKanFRTBaba et al., 2006JW4288Keio collection Baba et al., 2006
Strain, strain background (E. coli)AB1157dnaN-ypet FRTKanFRTReyes-Lamothe et al., 2010RRL190
Strain, strain background (E. coli)AB1157 ssb-ypet::KanReyes-Lamothe et al., 2008RRL32
Strain, strain background (E. coli)MG1655 PN25tetO::ruvCDef-gfp FRTXia et al., 2016SMR19425Doxycycline-inducible RDG cassette
Antibodyanti-RuvC (mouse monoclonal)Santa Cruz BiotechnologyCat# sc-53437, RRID:AB_630213IP (1:1000)
Recombinant DNA reagent pCP20Cherepanov and Wackernagel, 1995Flippase FLP ApR recombinase
Recombinant DNA reagentpJZ087Wang et al., 2019FRT-141x tetO GmR integration
Recombinant DNA reagentpKD46Datsenko and Wanner, 2000FRTKanFRT for RDG cassette
Recombinant DNA reagentpDM15Magnan and Bates, 2015aPnahG::tetR-yfp CmR expression
Sequence-based reagent P1This paperPCR primerGGTATATCTCCTTCTTAAAGTTAAACAAAATTATTTCTAGAAGGGTTATGCGTTGTTCCA
Sequence-based reagentP2This paperPCR primerGTTTCATGCTATGCCAAACGAGAATGATTATCAAATTCATGTGTAGGCTGGAGCTGCTTC
Sequence-based reagentP3This paperPCR primerCCCTAATTCGATGAAGATTCTTGCTCAATTGTTATCAGCGTGTAGGCTGGAGCTGCTTC
Sequence-based reagentP4This paperPCR primerAGACGTTTGGCTGATCGGCAAGGTGTTCTGGTCGGCGATTCCGGGGATCCGTCGACC
Commercial assay or kitNextera XT DNA Sample Preparation KitIlluminaCat #: FC-131-1024
Commercial assay or kitMiSeq Reagent Kit v3 (150-cycle) IlluminaCat #: MS-102-3001
Chemical compound, drugAnhydrotetracycline hydrochlorideSigma-AldrichCat #: 37919
Chemical compound, drugSodium salicylateSigma-AldrichCat #: S3007
Software, algorithmFocusCounterhttps://github.com/DavidBatesLab/Matlab-scripts.git; Joshi et al., 2013Matlab scriptCounting replisome foci
Software, algorithmFlowJoFlowJo LLCQuantifying flow cytometry histograms
Software, algorithmsequencingcompile.mhttps://github.com/DavidBatesLab/Matlab-scripts.gitMatlab scriptExtracting read coordinates from bam files
Software, algorithmsequencingcompile2.mhttps://github.com/DavidBatesLab/Matlab-scripts.gitMatlab scriptBinning reads into 1 kb bins

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