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.
