Journal: The Journal of Biological Chemistry
Article Title: TDP2/TTRAP Is the Major 5?-Tyrosyl DNA Phosphodiesterase Activity in Vertebrate Cells and Is Critical for Cellular Resistance to Topoisomerase II-induced DNA Damage *
Figure Lengend Snippet: The 5′-TDP activity of His-TDP2 prefers DSBs and liberates ligatable 5′ termini from DNA 5′-phosphotyrosine termini. A , purified recombinant human His-TDP2 (100 n m ) was incubated with 50 n m duplex substrate harboring a DSB with a 5′-phosphotyrosine ( P-Y ) terminus ( top ) in the absence or presence of the indicated concentrations of orthovanadate for 1 h at 37 °C and reaction products separated by denaturing PAGE and detected by autoradiography. Right , quantification of the fraction (%) of total labeled oligonucleotide converted to 5′-phosphate (19-mer) product is shown. Data are the average (± range) of two independent experiments. B , purified recombinant human His-TDP2 (100 n m ) was incubated with duplex substrate as above in the absence or presence of the indicated concentrations of BSA or phosphotyrosine-BSA. C , DNA substrates with a 5′-phosphotyrosine terminus present in single-stranded DNA ( top ), in a blunt-ended DNA duplex ( middle ), or in a nicked duplex ( bottom ), were incubated with the indicated concentrations of His-TDP2 for 1 h at 37 °C and reaction products analyzed as above. The middle graph is the quantification (% of total labeled oligonucleotide converted to 5′-P 19-mer product) of the above experiment, from three independent experiments mean (±S.E.). Asterisk denotes statistically significant difference ( p
Article Snippet: Reactions were carried out by incubating the indicated substrate (50 nm ) with whole cell protein extract (250 ng/ml) or the indicated concentration of recombinant human His-TDP2/TTRAP protein, which was expressed in Escherichia coli and prepared as described previously , in 25 mm HEPES, pH 8.0, 130 mm KCl, 10 mm MgCl2 , 1 mm DTT and in the presence or absence of orthovanadate, BSA, or phosphotyrosine-BSA (Sigma) as indicated at 37 °C for 60 min.
Techniques: Activity Assay, Purification, Recombinant, Incubation, Polyacrylamide Gel Electrophoresis, Autoradiography, Labeling