Shee, C, Cox, BD, Gu, F, Luengas, EM, Joshi, MC, Chiu, LY, Magnan, D, Halliday, JA, Frisch, RL, Gibson, JL, Nehring, RB, Do, HG, Hernandez, M, Li, L, Herman, C, Hastings, PJ, Bates, D, Harris, RS, Miller, KM and Rosenberg, SM (2013) Engineered proteins detect spontaneous DNA breakage in human and bacterial cells. Elife 2:e01222
Spontaneous DNA breaks instigate genomic changes that fuel cancer and evolution, yet direct quantification of double-strand breaks (DSBs) has been limited. Predominant sources of spontaneous DSBs remain elusive. We report synthetic technology for quantifying DSBs using fluorescent-protein fusions of double-strand DNA end-binding protein, Gam of bacteriophage Mu. In Escherichia coli GamGFP forms foci at chromosomal DSBs and pinpoints their subgenomic locations. Spontaneous DSBs occur mostly one per cell, and correspond with generations, supporting replicative models for spontaneous breakage, and providing the first true breakage rates. In mammalian cells GamGFP-labels laser-induced DSBs antagonized by end-binding protein Ku; co-localizes incompletely with DSB marker 53BP1 suggesting superior DSB-specificity; blocks resection; and demonstrates DNA breakage via APOBEC3A cytosine deaminase. We demonstrate directly that some spontaneous DSBs occur outside of S phase. The data illuminate spontaneous DNA breakage in E. coli and human cells and illustrate the versatility of fluorescent-Gam for interrogation of DSBs in living cells. DOI:http://dx.doi.org/10.7554/eLife.01222.001.
Animals; Bacteriophage mu/chemistry; Chromosomes, Bacterial/chemistry; Chromosomes, Bacterial/metabolism; Cytidine Deaminase/genetics; Cytidine Deaminase/metabolism; DNA/chemistry; DNA/metabolism; DNA Breaks, Double-Stranded; DNA Helicases/genetics; DNA Helicases/metabolism; DNA-Binding Proteins/genetics; DNA-Binding Proteins/metabolism; Escherichia coli/genetics; Escherichia coli/metabolism; Gene Expression Regulation; Genes, Reporter; Green Fluorescent Proteins/genetics; Green Fluorescent Proteins/metabolism; HeLa Cells; Humans; Intracellular Signaling Peptides and Proteins/genetics; Intracellular Signaling Peptides and Proteins/metabolism; Ku Autoantigen; Mice; Protein Engineering/methods; Proteins/genetics; Proteins/metabolism; Recombinant Fusion Proteins/genetics; Recombinant Fusion Proteins/metabolism; Synthetic Biology; Tumor Suppressor p53-Binding Protein 1; Viral Proteins/genetics; Viral Proteins/metabolism
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