TY - JOUR
T1 - Fluorescent fusions of the N protein of phage Mu label DNA damage in living cells
AU - Kotlajich, Matthew V.
AU - Xia, Jun
AU - Zhai, Yin
AU - Lin, Hsin Yu
AU - Bradley, Catherine C.
AU - Shen, Xi
AU - Mei, Qian
AU - Wang, Anthony Z.
AU - Lynn, Erica J.
AU - Shee, Chandan
AU - Chen, Li Tzu
AU - Li, Lei
AU - Miller, Kyle M.
AU - Herman, Christophe
AU - Hastings, P. J.
AU - Rosenberg, Susan M.
N1 - Publisher Copyright:
© 2018 The Authors
PY - 2018/12
Y1 - 2018/12
N2 - 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.
AB - 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.
KW - DNA damage
KW - Double-strand breaks
KW - Escherichia coli
KW - Phage Mu N protein
KW - Phleomycin
KW - RecBCD
KW - Single-cell analysis
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U2 - 10.1016/j.dnarep.2018.09.005
DO - 10.1016/j.dnarep.2018.09.005
M3 - Article
C2 - 30268364
AN - SCOPUS:85053887131
SN - 1568-7864
VL - 72
SP - 86
EP - 92
JO - DNA Repair
JF - DNA Repair
ER -