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. 2004 Feb;24(4):1595–1607. doi: 10.1128/MCB.24.4.1595-1607.2004

FIG. 5.

FIG. 5.

PARP-2 activity negatively regulates TRF2 DNA binding activity. (A) Schematic representation of the telomeric probe dsT4S1. The telomeric repeats are shown in bold. (B) Increasing amounts of either purified mPARP-2 (lanes 1 to 6) or purified hTRF2 (lanes 7 to 12) were incubated with the radiolabeled telomeric probe under binding conditions for 30 min at 20°C. Complexes were analyzed by electrophoresis at 4°C through a nondenaturing 1% agarose gel and phosphorimaging of the dried gel. The positions of free DNA and covalent complexes (mPARP-2-DNA and hTRF2-DNA) are indicated. (C) Purified mPARP-2 was incubated with the radiolabeled telomeric probe for various times as indicated. The complexes were analyzed on a nondenaturing 1% agarose gel as described for panel B. The positions of free DNA and covalent complexes (mPARP-2-DNA) are indicated. (D) Purified hTRF2 (100 nM) was preincubated with the radiolabeled telomeric probe for 10 min under binding conditions at 20°C, and purified mPARP-2 (100 nM) was subsequently added for 10 min (lanes 4 to 6), followed by the addition of the indicated antibodies (lanes 5 and 6). In lanes 1 to 3, the radiolabeled telomeric substrate was incubated with, respectively, no protein, hTRF2 (100 nM), and mPARP-2 (100 nM) for 30 min under binding conditions at 20°C. In lanes 7 to 10, purified hTRF2 (lanes 7 and 9) or purified mPARP-2 (lanes 8 and 10) was preincubated with the radiolabeled probe before the addition of the indicated antibodies. The binding products were analyzed on a nondenaturing 1% agarose gel as described for panel B. The positions of free DNA, covalent complexes (mPARP-2-hTRF2-DNA, hTRF2-DNA, and mPARP-2-DNA), and supershifts are indicated. (E) Purified hTRF2 (100 nM) was preincubated with the radiolabeled telomeric substrate for 10 min under binding conditions as described for panel D, and purified mPARP-2 (100 nM) was subsequently added for 10 min (lanes 4 to 10) followed by the addition of NAD+ for various times as indicated in the absence (lanes 4 to 9) or in the presence of 3-AB (lane 10). In lanes 1 to 3, the radiolabeled telomeric substrate was incubated with, respectively, no protein, mPARP-2 (100 nM), or hTRF2 (100 nM) for 30 min under binding conditions. The binding products were analyzed on a nondenaturing 1% agarose gel as described for panel B. The positions of free DNA, covalent complexes, and supershifts are indicated. Note that the migration of the mPARP-2-hTRF2-DNA complex was only slightly retarded compared to that of the hTRF2-DNA complex.