Research Paper Volume 1, Issue 3 pp 289—302

Figure 2. Concentration and time dependency of WRN exonuclease activity on telomeric substrates. (A) (left) 25 to 500 fmol of purified WRN were incubated with 5'-³²P-labeled, 3'-overhang telomeric DNA substrate at 37°C for 10 min. The reaction products were analyzed by 12% polyacrylamide-urea denaturing gel and autoradiography (lane 1 to 10, 25, 50, 100, 150, 200, 250, 300, 350, 400, 500 fmol of purified WRN; lane 11, DNA substrate. (Right) 200 fmol of purified WRN was incubated with 5'-³²P-labeled, 3'-overhang telomeric DNA substrate at 37°C from 0 to 10 min. The reaction products were analyzed by 12% polyacrylamide-urea denaturing gel and autoradiography (lane 1 to 11, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 min; lane 12, DNA substrate. (B) (left) 100 to 400 fmol of purified WRN were incubated with 5' ³²P-labeled, 3'-overhang telomeric DNA substrate in the absence or presence of 1.0 mM ATP or 1.0 mM Adenosine 5'-[γ-thio]triphosphate (ATPγS) at 37°C for 10 min. The reaction products were resolved by 12% polyacrylamide-urea denaturing gel and visualized by autoradiography (lane 1 to 4, 100, 200, 300, and 400 fmol of WRN without ATP; lane 5 to 8, 100, 200, 300, and 400 fmol of WRN in the presence of ATP, lane 9 to 12, 100, 200, 300, and 400 fmol of WRN in the presence of 1.0 mM ATPγS; lane 13, DNA substrate; lane 14, (TTAGGG) repeats molecular size markers. (Right) 100 to 400 fmol of purified WRN or WRN helicase mutant (K577M) were incubated with telomeric DNA substrates at 37°C for 10 min. The reaction products were analyzed by 12% polyacrylamide-urea denaturing gel and autoradiography (lane 1 to 4, 100, 200, 300, and 400 fmol of WRN; lane 5 to 8, 100, 200, 300, and 400 fmol of helicase mutant WRN; lane 9, DNA substrate; lane 10, (TTAGGG) repeats molecular size markers.