Research Paper Volume 1, Issue 3 pp 289—302

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

Figure 1. WRN exonuclease resects the 3' single-stranded overhang of telomeric DNA substrates.. (A) 100 to 400 fmol of purified recombinant wild-type WRN or exonuclease mutant WRN (WRN D82A) were incubated with 5'-32P-labeled, 15 nt 3'-overhang DNA substrates containing non-telomeric sequences (lanes 1-5) or telomeric (TTAGGG) repeats (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, non-telomeric DNA substrate; lane 6 to 9, 100, 200, 300, and 400 fmol of WRN; lane 10, telomeric DNA substrate; lane 11, (TTAGGG) repeats molecular size markers, lane 12 to 15, 100, 200, 300, and 400 fmol of exonuclease mutant WRN(D82A). (B) 100 to 400 fmol of purified recombinant WRN were incubated with 5'-32P-labeled, non-telomeric (lanes 1-5) or telomeric (lanes 6-10) DNA substrates with 27 nt 3'-overhang 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, non-telomeric DNA substrate; lane 6 to 9, 100, 200, 300, and 400 fmol of WRN; lane 10, telomeric DNA substrate, lane 11, (TTAGGG) repeats molecular size markers.