Method for detecting DNA strand breaks in mammalian cells using the Deinococcus radiodurans PprA protein.
In a previous study, we identified the novel protein PprA that plays a critical role in the radiation resistance of Deinococcus radiodurans. In this study, we focussed on the ability of PprA protein to recognize and bind to double-stranded DNA carrying strand breaks, and attempted to visualize radiation-induced DNA strand breaks in mammalian cultured cells by employing PprA protein using an immunofluorescence technique. Increased PprA protein binding to CHO-K1 nuclei immediately following irradiation suggests the protein is binding to DNA strand breaks. By altering the cell permeabilization conditions, PprA protein binding to CHO-K1 mitochondria, which is probably resulted from DNA strand break immediately following irradiation, was also detected. The method developed and detailed in this study will be useful in evaluating DNA damage responses in cultured cells, and could also be applicable to genotoxic tests in the environmental and pharmaceutical fields.
Satoh
Katsuya
K
Research Group for Biotechnology Development, Department of Ion-beam-applied Biology, Japan Atomic Energy Research Institute, 1233 Watanuki, Takasaki 370-1292, Japan. katsuya@taka.jaeri.go.jp
Wada
Seiichi
S
Kikuchi
Masahiro
M
Funayama
Tomoo
T
Narumi
Issay
I
Kobayashi
Yasuhiko
Y
D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism*; D016466:CHO Cells; D006224:Cricetinae; D004249:DNA Damage / Q000528:radiation effects*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D034301:Deinococcus / Q000235:genetics*; D004307:Dose-Response Relationship, Radiation; D005720:Gamma Rays; D008322:Mammals; D008928:Mitochondria / Q000502:physiology
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins