Research Paper Volume 1, Issue 3 pp 289—302

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

Figure 4. WRN exonuclease does not process telomeric DNA substrates with nucleotide substitutions within the 3' overhang sequence that alter the telomeric repeat unit.. (A) 100 to 400 fmol of purified WRN were incubated with 5'-32P-labeled telomeric DNA substrates with nucleotide substitutions within the 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, telomeric DNA substrate with 3' overhang CCC (69 to 71) substitution (substrate A); lane 6 to 9, 100, 200, 200, and 400 fmol of WRN; lane 10, telomeric DNA substrate with 3'overhang CCC (72 to 74) substitution (substrate B); lane 11 to 14, 100, 200, 300, and 400 fmol of WRN; lane 15, telomeric DNA substrate C. (B) 100 to 400 fmol of purified WRN were incubated with 5'-32P-labeled telomeric DNA substrates with either 18 nt (substrate A) or 15 nt (substrate B) 3' overhangs 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 A; lane 6 to 9, 100, 200, 300, and 400 fmol of WRN; lane 10, DNA substrate B.