Imaging mismatch repair and cellular responses to DNA damage in Bacillus subtilis.
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.
Klocko
Andrew D
AD
Department of Molecular, Cellular, and Developmental Biology, University of Michigan-Ann Arbor, USA.
Crafton
Kaleena M
KM
Walsh
Brian W
BW
Lenhart
Justin S
JS
Simmons
Lyle A
LA
D001412:Bacillus subtilis / Q000235:genetics*; D004249:DNA Damage; D053843:DNA Mismatch Repair; D004261:DNA Replication; D004269:DNA, Bacterial / Q000032:analysis* / Q000235:genetics; D008856:Microscopy, Fluorescence / Q000379:methods*
D004269:DNA, Bacterial