Research Paper Volume 1, Issue 3 pp 289—302

Sequence-specific processing of telomeric 3' overhangs by the Werner syndrome protein exonuclease activity

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.