Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis

Summary

A detailed protocol is described for imaging the real time formation of DNA repair complexes in Bacillus subtilis cells.

Abstract

Both prokaryotes and eukaryotes respond to DNA damage through a complex set of physiological changes. Alterations in gene expression, the redistribution of existing proteins, and the assembly of new protein complexes can be stimulated by a variety of DNA lesions and mismatched DNA base pairs. Fluorescence microscopy has been used as a powerful experimental tool for visualizing and quantifying these and other responses to DNA lesions and to monitor DNA replication status within the complex subcellular architecture of a living cell. Translational fusions between fluorescent reporter proteins and components of the DNA replication and repair machinery have been used to determine the cues that target DNA repair proteins to their cognate lesions in vivo and to understand how these proteins are organized within bacterial cells. In addition, transcriptional and translational fusions linked to DNA damage inducible promoters have revealed which cells within a population have activated genotoxic stress responses. In this review, we provide a detailed protocol for using fluorescence microscopy to image the assembly of DNA repair and DNA replication complexes in single bacterial cells. In particular, this work focuses on imaging mismatch repair proteins, homologous recombination, DNA replication and an SOS-inducible protein in Bacillus subtilis. All of the procedures described here are easily amenable for imaging protein complexes in a variety of bacterial species.

Protocol

Growing cultures of cells for microscopy

1. One or two days prior to imaging, prepare the B. subtilis strain containing the translational fusion protein you wish to visualize. Trial rounds of steps 1A and 1B will be necessary to determine which growth condition provides the best images of your strain. In most cases, cells should be imaged during exponential growth phase.

1. For some B. subtilis strains (in particular, strains that grow poorly due to the integration of the translational fusion between the gene of interest and GFP at its endogenous locus), initial growth for two days prior to imagin....

Discussion

Trial and error are required to find exposure conditions for the highest quality images for each strain; we find that 1 millisecond is appropriate for white light images, while exposures of 100 to 2000 ms are appropriate for GFP (FITC) and FM4-64 (TRITC) images. Exposure time will vary depending on the imaging equipment used. We recommend the use of one strain per 15-well microscope slide for the simplest imaging, as pad quality and diffusion of cells from the pad border could complicate strain differentiation if multipl.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Specific solution recipes1:
10x S750 salts 0.5 M MOPS 100 mM Ammonium Sulfate 50 mM Potassium Phosphate Monobasic Filter sterilize, and wrap S750 media in foil prior to storage
100x Metals 0.2 M MgCl2 70 mM CaCl2 5 mM MnCl2 0.1 mM ZnCl2 100 μg/mL Thiamine HCl 2 mM HCl 0.5 mM FeCl3* dH2O to final volume *FeCl3 should be added last, to prevent precipitation. After filter sterilization, wrap 100x Metal solution in foil.
S750 media 1x S750 salts 1x Metal 1% Glucose 0.1% Glutamate 40 μg/mL Tryptophan 40 μg/mL Phenylalanine distilled H2O to final volume
10x Spizizens (grams/L) 151.4 mM Ammonium Sulfate (20g/L) 803.8 mM Potassium Phosphate Monobasic (140g/L) 440.9 mM Potassium Phosphate Dibasic (60g/L) 34.0 mM Sodium Citrate (10g/L) 16.6 mM MgSO4 (2g/L) dH2O to final volume Filter sterilize