Research Paper Volume 1, Issue 3 pp 289—302

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

Figure 5. POT1 but not TRF2 inhibits processing of the 3' overhang of telomeric DNA substrates by WRN exonuclease.. (A) 200 to 800 fmol of purified TRF2 or TRF2ΔB were incubated with telomeric DNA substrates at room temperature for 20 min, then 200 fmol of WRN was added into the reaction to incubate at 37ºC for 15 min. The products were analyzed by 12% polyacrylamide-urea denaturing gel and autoradiography (lane 1, 200 fmol of WRN; lane 2 to 5, 200, 400, 600, 800 fmol of TRF2 and 200 fmol of WRN; lane 6, 200 fmol of WRN; lane 7 to 10, 200, 400, 600, 800 fmol of TRF2ΔB and 200fmol of WRN; lane 11, DNA substrate. (B)200 to 800 fmol of purified POT1 or POT1Δ1-140 were incubated with telomeric DNA substrates at room temperature for 20 min, then 200 fmol of WRN was added into the reaction to incubate at 37ºC for 15 min. The products were analyzed by 12% polyacrylamide-urea denaturing gel and autoradiography (lane 1, 200 fmol of WRN; lane 2 to 5, 200, 400, 600, 800 fmol POT1 and 200 fmol of WRN; lane 6, 200 fmol of WRN; lane 7 to 10, 200, 400, 600, 800 fmol POT1Δ1-140 and 200 fmol of WRN; lane 11, DNA substrate). (C) 200 to 800 fmol of purified POT1 or POT1Δ1-140 were incubated with telomeric substrates with 27 nt 3' overhang at room temperature for 20 min, then 200 fmol of WRN was added into the reaction to incubate at 37ºC for 15 min. The products were analyzed by 12% polyacrylamide-urea denaturing gel and autoradiography (lane 1, 200 fmol of WRN; lane 2 to 5, 200, 400, 600, 800 fmol of POT1 and 200 fmol of WRN; lane 6, 200 fmol of WRN;lane 7 to 10, 200, 400, 600, 800 fmol of POT1Δ140 and 200 fmol of WRN; lane 11, 89-mer telomeric oligo, lane 12 77-mer telomeric oligo. (D) Silver stained SDS-PAGE gel showing the purity of purified recombinant TRF2, TRF2ΔB, Pot1, and POT1Δ1-140 used in the exonuclease assays.