Real-time monitoring of uracil removal by uracil-DNA glycosylase using fluorescent resonance energy transfer probes.
As a highly conserved damage repair protein, uracil-DNA glycosylase (UDG) mainly catalyzes the excision of uracil from DNA to sustain the genome integrity. Here a novel method for monitoring the uracil removal in real time is introduced. Double-stranded DNA probes modified with uracil residues that can occur in fluorescent resonance energy transfer (FRET) were used as substrates and detecting probes in a homogeneous solution. This method not only overcame the drawbacks of traditional radioactive assays, such as discontinuity and being time-consuming and complicated, but also was used to accurately determine the kinetic constant of UDG. The limit of detection of UDG was 0.033 U/ml. The KM and Kcat were 0.11 microM and 4 s(-1), respectively. In addition, the method was applied to investigate the influence of chemical drugs on UDG activity. The results showed that 10 mM fluorouracil (5-FU) and gentamicin are inhibitors to UDG. The in vitro detection of UDG in A549 cells showed that the activity of UDG was four times greater after the cells were treated with cisplatin. These results showed that this method can monitor uracil removal in real time and conveniently assay UDG activity with ultrasensitivity and excellent specificity in the homogeneous solution. This method is also amenable to high-throughput drug screening in vitro.
Liu
Bin
B
Institute of Life Science and Biotechnology, College of Chemistry and Chemical Engineering, Hunan University, and Key Laboratory for Bio-Nanotechnology and Molecular Engineering of Hunan Province, Changsha 410082, People's Republic of China.
Yang
Xiaohai
X
Wang
Kemin
K
Tan
Weihong
W
Li
Huimin
H
Tang
Hongxing
H
D001483:Base Sequence; D002384:Catalysis / Q000187:drug effects; D045744:Cell Line, Tumor; D002474:Cell-Free System / Q000378:metabolism; D002945:Cisplatin / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005472:Fluorouracil / Q000494:pharmacology; D005839:Gentamicins / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008954:Models, Biological; D015203:Reproducibility of Results; D014498:Uracil / Q000737:chemistry / Q000378:metabolism*; D051981:Uracil-DNA Glycosidase / Q000037:antagonists & inhibitors / Q000378:metabolism*
D005839:Gentamicins; D014498:Uracil; D051981:Uracil-DNA Glycosidase; D002945:Cisplatin; D005472:Fluorouracil