title: Real time monitoring of junction ribonuclease activity of RNase H using chimeric molecular beacons.
abstract: As a highly conserved damage repair protein, except for hydrolysis of DNA-RNA heteroduplex endonucleolytically, RNase H can cleave RNA-DNA junctions in Okazaki fragment processing through its junction ribonuclease (JRNase) activity. We report here a real time fluorescence method for detecting JRNase activity of RNase H with high accuracy by applying chimeric molecular beacons as substrates. The detection limit of E. coli RNase H is 0.5 U ml(-1). The Km and kcat are 20 nM and 0.6 s(-1), respectively. In addition, we used the method to investigate the effect of chemical drugs on the enzyme and found that both penicillin and streptomycin sulfate inhibit its activity with the IC50 values of 0.2 and 0.07 mM, respectively. Finally, we applied the method to reliably detect the JRNase level in tumor cells. In summary, these data indicate that the simple, rapid and sensitive method can be hopefully applied for high-throughput detection of samples and drug screening in vitro.
authors.affiliation: College of Biology, Hunan University, Changsha, Hunan 410082, People's Republic of China. binliu2001@hotmail.com
authors.forename: Bin
authors.initials: B
authors.lastname: Liu
authors.forename: Dan
authors.initials: D
authors.lastname: Xiang
authors.forename: Ying
authors.initials: Y
authors.lastname: Long
authors.forename: Chunyi
authors.initials: C
authors.lastname: Tong
mesh_terms: D001483:Base Sequence; D045744:Cell Line, Tumor; D004247:DNA / Q000235:genetics / Q000378:metabolism; D057075:Enzyme Assays / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology; D006801:Humans; D007700:Kinetics; D015345:Oligonucleotide Probes / Q000235:genetics / Q000378:metabolism*; D016914:Ribonuclease H / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013997:Time Factors
chemical_list: D004791:Enzyme Inhibitors; D015345:Oligonucleotide Probes; D004247:DNA; D016914:Ribonuclease H