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 ...

• Main Authors: Keck, James, McKenzie, Aidan, Henry, Camille, Myers, Kevin, Place, Michael
• Format: Dataset
• Language: English
• Published: Dryad 2022
• Subjects: FOS Biological sciences; MuGam-GFP; Fluorescence microscopy; bright-field image; DNA double-strand break; DNA replication restart; Orca
• Online Access: https://dx.doi.org/10.5061/dryad.547d7wmbx; https://datadryad.org/stash/dataset/doi:10.5061/dryad.547d7wmbx

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 ... : An E. coli strain carrying MuGam-GFP (SMR14334) was derivatized to carry the sulB103 allele (wt) 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 OD600 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). ...