Fig 1. p21 increases the level of PARP-1 crosslinking to nick-containing BER DNA intermediates.

(A) Schematic diagram showing the structure of p21 full length, with CDK and PCNA binding domains, and of the C terminal fragment (p21 Cter) lacking the first N-terminal 74 aa. (B) Crosslinking of PARP1 of the nuclear extract in the presence of p21 or p21Cter. [³²P]-labeled 0.1 μM DNA*pF (lanes 1–3, 7) or DNA*p (lanes 4–6, 8) was incubated with bovine testis nuclear extract (BTNE) proteins (lanes 1–3, 4–6) in the presence of 1.2 μM p21 (lanes 2, 5) or p21Cter (lanes 3, 6). Loading control: DNA*pF, and DNA*p incubated with recombinant PARP-1 (lanes 7, 8). (C) Crosslinking of recombinant PARP1 in the presence of p21 or p21Cter. [³²P]-labeled 0.1 μM DNA*pF (lanes 1–4) or DNA*p (lanes 5–8) was incubated with recombinant PARP-1 in the presence of p21 (lanes 3, 7) or p21Cter (lanes 4, 8). Then the mixtures were UV irradiated (lanes 2–4 and 6–8). Free DNA probe is also shown. The products were separated on 10% SDS-PAGE and analyzed by PhosphorImaging. The reaction conditions and analysis of the reaction products are described in “Materials and Methods”. The nucleotide sequence and structure of (DNA*pF and DNA*p) is reported (Table 1). (D) Diagram showing the quantitative analysis results shown in panel C. The crosslinking of PARP-1 was defined as the percentage (%) of photoreactive DNA, which formed covalent adducts with PARP-1. Error bars represent relative mean ± SD, n = 4. Asterisks denote statistically significant difference (*, P<0.05, ns, not significant; t-test).