FIG. 6.
Poly(ADP-ribose) inhibits the DNA binding activity of hTRF2. (A) Purified hTRF2 (30 nM) was incubated with the radiolabeled telomeric probe and increasing amounts of poly(ADP-ribose) as indicated for 30 min at 20°C under binding conditions as described in the legend for Fig. 5. The binding products were analyzed by autoradiography after nondenaturing 1% agarose gel electrophoresis. The positions of free DNA and covalent complexes (hTRF2-DNA) are indicated. (B) Poly(ADP-ribose) binds to purified recombinant hTRF2. Upper panel: 1 μg of purified recombinant hTRF2, hXRCC1, or mPARP-2 was spotted onto nitrocellulose and incubated with [32P]poly(ADP-ribose) as described in Materials and Methods. Lower panel: the amount of protein loaded was controlled by Coomassie staining. (C) Poly(ADP-ribose) binds to the myb domain of hTRF2. GST (lane 1), GST-hXRCC1141-572 (lane 2), GST-hTRF2 (lane 3), and GST-tagged deletion mutants of hTRF2 (lanes 4 to 6) were expressed in Cos1 cells, extracted by GST pull down, separated on SDS-12% PAGE, blotted onto nitrocellulose membrane, renatured, and incubated with [32P]poly(ADP-ribose) as described for panel B. Lane 7, hTRF2 (1 μg). Upper panel: autoradiography. Lower panel: subsequently, fusion proteins were analyzed by Western blotting with anti-GST.