Fluorescent fusions of the N protein of phage Mu label DNA damage in living cells

DNA Repair (Amst). 2018 Dec;72:86-92. doi: 10.1016/j.dnarep.2018.09.005. Epub 2018 Sep 14.

Abstract

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.

Keywords: DNA damage; Double-strand breaks; Escherichia coli; Phage Mu N protein; Phleomycin; RecBCD; Single-cell analysis.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacteriophage mu / genetics*
  • Bacteriophage mu / metabolism*
  • DNA Breaks, Double-Stranded
  • DNA Damage*
  • Escherichia coli / cytology
  • Exonucleases / metabolism
  • Fluorescent Dyes / metabolism*
  • Phleomycins / metabolism
  • Viral Regulatory and Accessory Proteins / metabolism*

Substances

  • Fluorescent Dyes
  • Phleomycins
  • Viral Regulatory and Accessory Proteins
  • Exonucleases