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 …
(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, …
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).
(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) …
(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. …
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 …
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. …
(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 …
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 …
(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) …
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 …
Cumulative curves of DNA histograms (Figure 4—figure supplement 1) with 90-min time point overlayed (gray).
(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…
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 …
(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 …
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), …
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.
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Strain, strain background (Escherichia coli) | AB1157 dnaN-mCherry FRTKanFRT | Moolman et al., 2014 | BN1682 | |
Strain, strain background (E. coli) | AB1157 dnaN-ypet FRT | This paper | DB1568 | RRL190 flipped to KmS w/pCP20 |
Strain, strain background (E. coli) | MG1655 141x tetO array @ lac, pDM15 (+1.10 Mb block) | Joshi et al., 2011 | DB2146 | |
Strain, strain background (E. coli) | MG1655 141x tetO array @ glnA, pDM15 (+0.13 Mb block) | Joshi et al., 2011 | DB2185 | |
Strain, strain background (E. coli) | MG1655 141x tetO array @ dnaB, pDM15 (+0.34 Mb block) | Joshi et al., 2011 | DB2193 | |
Strain, strain background (E. coli) | MG1655 ΔrecB FRTKanFRT | This paper | DB2257 | MG1655 × P1.JW2788 |
Strain, strain background (E. coli) | MG1655 141x tetO array @ gln, ΔrecB FRTKanFRT, pDM15 (+0.13 Mb block) | This paper | DB2403 | DB2185 × P1.JW2788 |
Strain, strain background (E. coli) | MG1655 141x tetO array @ dnaB, ΔrecB FRTKanFRT, pDM15 (+0.34 Mb block) | This paper | DB2722 | DB2193 × P1.JW2788 |
Strain, strain background (E. coli) | MG1655 141x tetO array @ lac, ΔrecB FRTKanFRT, pDM15 (+1.10 Mb block) | This paper | DB2725 | DB2146 × P1.JW2788 |
Strain, strain background (E. coli) | MG1655 dnaN-mCherry FRTKanFRT | This paper | DB2943 | MG1655 × P1.BN1682 |
Strain, strain background (E. coli) | MG1655 λcIts857 PR::ruvCDef-gfp FRTKanFRT (42°C inducible RDG cassette) | This paper | DB2945 | SMR19379 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 paper | DB2954 | DB2185 × P1.DB2945 |
Strain, strain background (E. coli) | MG1655 141x tetO array @ dnaB, λcIts857 PR::ruvCDef-gfp FRTKanFRT, pDM15 (+0.34 Mb block, RDG) | This paper | DB2956 | DB2193 × P1.DB2945 |
Strain, strain background (E. coli) | MG1655 dnaN-mCherry FRT | This paper | DB2962 | DB2943 flipped to KmS w/pCP20 |
Strain, strain background (E. coli) | MG1655 141x tetO array @ glnA, pDM15 (+0.13 Mb block) dnaN-mCherry FRTKanFRT | This paper | DB2970 | DB2185 × P1.BN1682 |
Strain, strain background (E. coli) | MG1655 141x tetO array @ glnA, pDM15 (+0.13 Mb block) dnaN-mCherry FRT | This paper | DB2972 | DB2970 flipped to KmS w/pCP20 |
Strain, strain background (E. coli) | BW25113 yjiC::141x tetO array FRT | This paper | DB3094 | JW4288 × pJZ087 & pCP20 |
Strain, strain background (E. coli) | BW25113 yjbL::141x tetO array FRT | This paper | DB3096 | JW4007 × pJZ087 & pCP20 |
Strain, strain background (E. coli) | MG1655 141x tetO array @ lac, λcIts857 PR::ruvCDef-gfp FRTKanFRT, pDM15 (+1.10 Mb block, RDG) | This paper | DB3124 | DB2146 × P1.DB2945 |
Strain, strain background (E. coli) | MG1655 yjiC::141x tetO array FRT | This paper | DB3191 | MG16556 × P1.DB3094 |
Strain, strain background (E. coli) | MG1655 yjbL::141x tetO array FRT | This paper | DB3193 | MG1655 × P1.DB3096 |
Strain, strain background (E. coli) | MG1655 yjiC::141x tetO array FRT, pDM15 (+0.63 Mb block) | This paper | DB3195 | DB3191 × pDM15 |
Strain, strain background (E. coli) | MG1655 yjbL::141x tetO array FRT pDM15 (+0.32 Mb block) | This paper | DB3198 | DB3193 × pDM15 |
Strain, strain background (E. coli) | BW25113 yhgN::141x tetO array FRT | This paper | DB3212 | JW3397 × pJZ087 & pCP20 |
Strain, strain background (E. coli) | MG1655 yhgN::141x tetO array FRT | This paper | DB3214 | MG1655 × P1.3212 |
Strain, strain background (E. coli) | MG1655 yhgN::141x tetO array FRT, pDM15 (−0.35 Mb block) | This paper | DB3216 | DB3214 × pDM15 |
Strain, strain background (E. coli) | BW25113 yjgZ::141x tetO array FRT | This paper | DB3258 | JW4236 × pJZ087 & pCP20 |
Strain, strain background (E. coli) | MG1655 yjgZ::141x tetO array FRT | This paper | DB3262 | MG1655 × P1.DB3258 |
Strain, strain background (E. coli) | MG1655 yjgZ::141x tetO array FRT, pDM15 (+0.58 Mb block) | This paper | DB3264 | DB3262 × pDM15 |
Strain, strain background (E. coli) | BW25113 ΔrecB FRTKanFRT | Baba et al., 2006 | JW2788 | Keio collection |
Strain, strain background (E. coli) | BW25113 ∆yhgN FRTKanFRT | Baba et al., 2006 | JW3397 | Keio collection |
Strain, strain background (E. coli) | BW25113 ∆yjbL FRTKanFRT | Baba et al., 2006 | JW4007 | Keio collection |
Strain, strain background (E. coli) | BW25113 ∆yjgZ FRTKanFRT | Baba et al., 2006 | JW4236 | Keio collection |
Strain, strain background (E. coli) | BW25113 ∆yjiC FRTKanFRT | Baba et al., 2006 | JW4288 | Keio collection Baba et al., 2006 |
Strain, strain background (E. coli) | AB1157dnaN-ypet FRTKanFRT | Reyes-Lamothe et al., 2010 | RRL190 | |
Strain, strain background (E. coli) | AB1157 ssb-ypet::Kan | Reyes-Lamothe et al., 2008 | RRL32 | |
Strain, strain background (E. coli) | MG1655 PN25tetO::ruvCDef-gfp FRT | Xia et al., 2016 | SMR19425 | Doxycycline-inducible RDG cassette |
Antibody | anti-RuvC (mouse monoclonal) | Santa Cruz Biotechnology | Cat# sc-53437, RRID:AB_630213 | IP (1:1000) |
Recombinant DNA reagent | pCP20 | Cherepanov and Wackernagel, 1995 | Flippase FLP ApR recombinase | |
Recombinant DNA reagent | pJZ087 | Wang et al., 2019 | FRT-141x tetO GmR integration | |
Recombinant DNA reagent | pKD46 | Datsenko and Wanner, 2000 | FRTKanFRT for RDG cassette | |
Recombinant DNA reagent | pDM15 | Magnan and Bates, 2015a | PnahG::tetR-yfp CmR expression | |
Sequence-based reagent | P1 | This paper | PCR primer | GGTATATCTCCTTCTTAAAGTTAAACAAAATTATTTCTAGAAGGGTTATGCGTTGTTCCA |
Sequence-based reagent | P2 | This paper | PCR primer | GTTTCATGCTATGCCAAACGAGAATGATTATCAAATTCATGTGTAGGCTGGAGCTGCTTC |
Sequence-based reagent | P3 | This paper | PCR primer | CCCTAATTCGATGAAGATTCTTGCTCAATTGTTATCAGCGTGTAGGCTGGAGCTGCTTC |
Sequence-based reagent | P4 | This paper | PCR primer | AGACGTTTGGCTGATCGGCAAGGTGTTCTGGTCGGCGATTCCGGGGATCCGTCGACC |
Commercial assay or kit | Nextera XT DNA Sample Preparation Kit | Illumina | Cat #: FC-131-1024 | |
Commercial assay or kit | MiSeq Reagent Kit v3 (150-cycle) | Illumina | Cat #: MS-102-3001 | |
Chemical compound, drug | Anhydrotetracycline hydrochloride | Sigma-Aldrich | Cat #: 37919 | |
Chemical compound, drug | Sodium salicylate | Sigma-Aldrich | Cat #: S3007 | |
Software, algorithm | FocusCounter | https://github.com/DavidBatesLab/Matlab-scripts.git; Joshi et al., 2013 | Matlab script | Counting replisome foci |
Software, algorithm | FlowJo | FlowJo LLC | Quantifying flow cytometry histograms | |
Software, algorithm | sequencingcompile.m | https://github.com/DavidBatesLab/Matlab-scripts.git | Matlab script | Extracting read coordinates from bam files |
Software, algorithm | sequencingcompile2.m | https://github.com/DavidBatesLab/Matlab-scripts.git | Matlab script | Binning reads into 1 kb bins |