DOI: 10.5061/dryad.547d7wmbx
title: Microscopy data from: Identification of genetic interactions with priB links the PriA/PriB DNA replication restart pathway to double-strand DNA break repair in Escherichia coli

description: Collisions between DNA replication complexes (replisomes) and impediments
 such as damaged DNA or proteins tightly bound to the chromosome lead to
 premature dissociation of replisomes at least once per cell cycle in
 Escherichia coli. Left unrepaired, these events produce incompletely
 replicated chromosomes that cannot be properly partitioned into daughter
 cells. DNA replication restart, the process that reloads replisomes at
 prematurely terminated sites, is therefore essential in E. coli and other
 bacteria. Three replication restart pathways have been identified in E.
 coli: PriA/PriB, PriA/PriC, and PriC/Rep. A limited number of genetic
 interactions between replication restart and other genome maintenance
 pathways have been defined, but a systematic study placing replication
 restart reactions in a broader cellular context has not been performed. We
 have utilized transposon insertion sequencing to identify new genetic
 interactions between DNA replication restart pathways and other cellular
 systems. Known genetic interactors with the priB replication restart gene
 (uniquely involved in the PriA/PriB pathway) were confirmed and several
 novel priB interactions were discovered. Far fewer connections were found
 with the PriA/PriC or PriC/Rep pathways, suggesting a primacy role for the
 PriA/PriB pathway in E. coli. Targeted genetic and imaging-based
 experiments with priB and its genetic partners revealed significant
 double-strand DNA break (DSB) accumulation in strains with mutations in
 dam, rep, rdgC, lexA, or polA. Modulating the activity of the RecA
 recombinase partially suppressed the detrimental effects of rdgC or lexA
 mutations in ΔpriB cells. Taken together, our results highlight roles for
 several genes in DSB homeostasis and define a genetic network that
 facilitates DNA repair/processing upstream of PriA/PriB-mediated DNA
 replication restart in E. coli.
description: An <em>E. coli</em> strain carrying MuGam-GFP
 (SMR14334) was derivatized to carry the<em> sulB103</em>
 allele (<em>wt</em>) before P1 transduction deleted other
 genes of interest. Saturated cultures were diluted 100x and grown in LB
 for 30 min at 37 °C to enter the early exponential phase. MuGam-GFP
 expression was then induced at 100 ng/mL doxycycline and growth continued
 for an additional 2.5 hr at 37 °C. Cells were pelleted and resuspended in
 1x PBS buffer (to OD<sub>600</sub> of 1.0) and placed on ice.
 About 15 min prior to imaging, cell membrane stain FM 4-64 (5 mM) was
 added, and 2-3 µL of cells were sandwiched between a 24x50 mM, No. 1.5
 coverslip (Azer Scientific) and a 1.5% agarose pad. All cells were imaged
 at room temperature with a motorized inverted Nikon Ti-eclipse N-STORM
 microscope equipped with a 100x objective and ORCA Flash 4.0 digital CMOS
 C13440 (Hamatsu). Imaging was performed using NIS-Elements software with
 the microscope in epifluorescence mode. Cells were first imaged in the
 brightfield (4.5 V, 100 ms exposure). Visualization of the cell membranes
 was performed in the DsRed channel to ensure the focusing (4.5 V, 50 ms
 exposure) and then MuGam-GFP was imaged in the GFP channel (4.5 V, 50 ms
 exposure). Growth, preparation, and imaging were performed for each strain
 in biological triplicate. Analysis of cell features was
 performed with Fiji software (ImageJ) equipped with plugins as described
 previously: Single Molecule Biophysics
 (https://github.com/SingleMolecule/smb-plugins) and MicrobeJ. Briefly, the
 nd2 raw images for each strain (4 to 8 per replicate with a maximum
 difference of 2 images within triplicate) were concatenated together by
 channels. The image processing of each channel was carried out the same
 way and uniformly throughout the field of view. The scale of all images
 was corrected to fit the Hamamatsu camera scale. The brightfield and DsRed
 image stacks were auto-scaled while the GFP images were processed with
 discoidal averaging of 1-5 and intensity scale set at 0-300. Both
 brightfield and DsRed channels were cleaned by running a Bandpass filter
 10_2 with autoscale 5, a rolling sliding stack of 10, and an enhance
 contrast of 0.1. Channel stacks were converted to 8 bits before analysis
 in MicrobeJ. For the analysis, hyperstacks combining only the FM 4-64 and
 GFP channels were generated in MicrobeJ. From these hyperstacks, cell
 outlines were detected in the DsRed channel using the default method with
 a threshold of +25. Within identified cells, GFP foci were detected using
 the maxima features as foci with a Gaussian fit constraint. The exact
 setup used to identify bacteria and MuGam-GFP foci in MicrobeJ is
 available (Final Bacteria setup 1_5 foci 90) as a .xml file. After
 automatic detection, cells were manually sorted to remove poorly fitting
 outlines or outlines fitting to cells out of focus. Cell features analysis
 acquired with MicrobeJ (cell ID, cell length, number of foci per cell,
 foci intensity, and size) were exported as .csv files. Plots and
 statistical analysis were generated and performed with GraphPad Prism
 software. At least 650 single cells were analyzed for each
 condition. 
description: Microsoft Excel; Fiji (equipped with single molecule and MicrobeJ
 plugins); GraphPad Prism.