Inducing a Site Specific Replication Blockage in <i>E. coli</i> Using a Fluorescent Repressor Operator System

Abstract

Obstacles present on DNA, including tightly-bound proteins and various lesions, can severely inhibit the progression of the cell's replication machinery. The stalling of a replisome can lead to its dissociation from the chromosome, either in part or its entirety, leading to the collapse of the replication fork. The recovery from this collapse is a necessity for the cell to accurately complete chromosomal duplication and subsequently divide. Therefore, when the collapse occurs, the cell has evolved diverse mechanisms that take place to restore the DNA fork and allow replication to be completed with high fidelity. Previously, these replication repair pathways in bacteria have been studied using UV damage, which has the disadvantage of not being localized to a known site. This manuscript describes a system utilizing a Fluorescence Repressor Operator System (FROS) to create a site-specific protein block that can induce the stalling and collapse of replication forks in Escherichia coli. Protocols detail how the status of replication can be visualized in single living cells using fluorescence microscopy and DNA replication intermediates can be analyzed by 2-dimensional agarose gel electrophoresis. Temperature sensitive mutants of replisome components (e.g. DnaBts) can be incorporated into the system to induce a synchronous collapse of the replication forks. Furthermore, the roles of the recombination proteins and helicases that are involved in these processes can be studied using genetic knockouts within this system.

Replication fork collapse at a protein‐DNA roadblock leads to fork reversal, promoted by the RecQ helicase

“…Production of the fluorescent repressor TetR‐YFP was induced by addition of 0.1% (w/v) arabinose once cells had reached at least OD 600nm = 0.05. Cells were then incubated for 90 min and examined under a fluorescence microscope as described previously (Mettrick et al , ) to confirm the extent of replication blockage across the cell population. A minimum of 200 cells were assessed and foci counted.…”

“…Samples of cells were taken at the indicated time points and the DNA prepared as previously outlined (Mettrick and Grainge, ; Mettrick et al , ). DNA was digested with either EcoRI (array region) or HincII (region upstream of the array).…”

Replication fork collapse at a protein-DNA roadblock leads to fork reversal, promoted by the RecQ helicase

“…Production of the fluorescent repressor TetRYFP was induced by addition of 0.1% (w/v) arabinose once cells had reached at least OD 600nm = 0.05. Cells were then incubated for 90 minutes and examined under a fluorescence microscope as described previously (70) to confirm the extent of replication blockage across the cell population. A minimum of 200 cells were assessed and foci counted.…”

“…Samples of cells were taken at the indicated time points and the DNA prepared as previously outlined (44, 70). DNA was digested with either EcoRI (array region) or HincII (region upstream of the array).…”

“…DNA was digested with either EcoRI (array region) or HincII (region upstream of the array). 2-D gel conditions and Southern hybridisation were as described by (70). Statistical analysis was determined using the Student’s t test.…”