Research Paper Volume 1, Issue 3 pp 289—302

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

Figure 3. Both single- and double-stranded telomeric DNA sequences are required for the processing of the 3' overhang by WRN exonuclease.. (A) 100 to 400 fmol of purified WRN were incubated with 5'-32P-labeled, 3'-overhang DNA substrate with (CCCAAT) repeats sequence (lanes 1-5) and telomeric DNA substrate (lanes 6-10) 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, (CCCAAT) repeat DNA substrate; lane 6 to 9, 100, 200, 300, and 400 fmol of WRN; lane 10, telomeric DNA substrate. (B) 100 to 400 fmol of purified WRN were incubated with 5'-32P-labeled DNA substrate with 3' of non-telomeric overhang (lanes 1-5) and telomeric DNA substrate (lanes 6-10) 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, telomeric DNA substrate with non-telomeric overnhang; lane 6 to 9, 100, 200, 300, and 400 fmol of WRN; lane 10, telomeric DNA substrate. (C) 100 to 400 fmol of purified WRN were incubated with 5'-32P-labeled, 3'-overhang DNA substrate with double-stranded non-telomeric sequence (lanes 1-5) and telomeric DNA substrate (lanes 6-10) 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, DNA substrate with double-stranded non-telomeric DNA sequence; lane 6 to 9, 100, 200, 300, and 400 fmol of WRN; lane 10, telomeric DNA substrate.