The N protein of phage Mu was indicated from studies in Escherichia coli to hold linear Mu chromosomes in a circular conformation by non-covalent association， and thus suggested potentially to bind DNA double-stranded ends. Because of its role in association with linear Mu DNA， we tested whether fluorescent-protein fusions to N might provide a useful tool for labeling DNA damage including double-strand break (DSB) ends in single cells. We compared N-GFP with a biochemically well documented DSB-end binding protein， the Gam protein of phage Mu， also fused to GFP. We find that N-GFP produced in live E. coli forms foci in response to DNA damage induced by radiomimetic drug phleomycin， indicating that it l... More
The N protein of phage Mu was indicated from studies in Escherichia coli to hold linear Mu chromosomes in a circular conformation by non-covalent association， and thus suggested potentially to bind DNA double-stranded ends. Because of its role in association with linear Mu DNA， we tested whether fluorescent-protein fusions to N might provide a useful tool for labeling DNA damage including double-strand break (DSB) ends in single cells. We compared N-GFP with a biochemically well documented DSB-end binding protein， the Gam protein of phage Mu， also fused to GFP. We find that N-GFP produced in live E. coli forms foci in response to DNA damage induced by radiomimetic drug phleomycin， indicating that it labels damaged DNA. N-GFP also labels specific DSBs created enzymatically by I-SceI double-strand endonuclease， and by X-rays， with the numbers of foci corresponding with the numbers of DSBs generated， indicating DSB labeling. However， whereas N-GFP forms about half as many foci as GamGFP with phleomycin， its labeling of I-SceI- and X-ray-induced DSBs is far less efficient than that of GamGFP. The data imply that N-GFP binds and labels DNA damage including DSBs， but may additionally label phleomycin-induced non-DSB damage， with which DSB-specific GamGFP does not interact. The data indicate that N-GFP labels DNA damage， and may be useful for general， not DSB-specific， DNA-damage detection.
