Poly(ADP-ribose) contributes to an association between poly(ADP-ribose) polymerase-1 and xeroderma pigmentosum complementation group A in nucleotide excision repair

Abstract

Exposure to ultraviolet radiation (UVR) promotes the formation of UVR-induced, DNA helix distorting photolesions such as (6-4) pyrimidine-pyrimidone photoproducts and cyclobutane pyrimidine dimers. Effective repair of such lesions by the nucleotide excision repair (NER) pathway is required to prevent DNA mutations and chromosome aberrations. Poly(ADP-ribose) polymerase-1 (PARP-1) is a zinc finger protein with well documented involvement in base excision repair. PARP-1 is activated in response to DNA damage and catalyzes the formation of poly(ADP-ribose) subunits that assist in the assembly of DNA repair proteins at sites of damage. In this study, we present evidence for PARP-1 contributions to NER, extending the knowledge of PARP-1 function in DNA repair beyond the established role in base excision repair. Silencing the PARP-1 protein or inhibiting PARP activity leads to retention of UVR-induced photolesions. PARP activation following UVR exposure promotes association between PARP-1 and XPA, a central protein in NER. Administration of PARP inhibitors confirms that poly(ADP-ribose) facilitates PARP-1 association with XPA in whole cell extracts, in isolated chromatin complexes, and in vitro. Furthermore, inhibition of PARP activity decreases UVR-stimulated XPA chromatin association, illustrating that these relationships occur in a meaningful context for NER. These results provide a mechanistic link for PARP activity in the repair of UVR-induced photoproducts.

FIGURE 1.

Effects of PARP activity on retention of UVR-induced photoproducts. A, representative Western blot comparing PARP-1 protein in HaCaT and PARP-1 HuSH cells. GAPDH is used as a loading control. B, quantification of A by densitometry. PARP-1 intensity was normalized to GAPDH. The data are presented as the means ± S.E., n = 3. C, quantification of PAR Western blots by densitometry in HaCaT and PARP-1 HuSH cells following UVR exposure. The data are presented as the means ± S.E., n = 3. NT = not treated. D, HaCaT cells and PARP-1 HuSH cells were exposed to a single dose of ssUVR (3 kJ/m²) and collected at various times postexposure. Immunofluorescence was used to obtain images of 6-4 PPs. Initial UVR-induced photoproducts did not differ between HaCaT and PARP-1 HuSH cells. E, fluorescence intensity obtained from images in D. Open triangles, HaCaT; closed circles, PARP-1 HuSH. Intensities were normalized to NT sample. The data are presented as the means ± S.E., n = 4. F and G, slot blot was performed on DNA extracted from HaCaT and PARP-1 HuSH cells. Quantification of lesion intensity was done by densitometry. The intensities were normalized to NT. F, intensity of 6-4 PPs formation. The data are presented as the means ± S.E., n = 3. G, intensity of CPD formation. The data are presented as the means ± S.E., n = 3. *, p < 0.05; **, p < 0.01; ***, p < 0.001. Scale bar, 50 μm.

FIGURE 2.

Inhibition of PARP activity results in retention of UVR-induced lesions. HaCaT cells were pre-exposed to a PARP inhibitor, DPQ, 30 min prior to a single dose of ssUVR (3 kJ/m²) or exposed to UVR alone and collected at the indicated times postexposure. A, quantification of PAR Western blots by densitometry. The data are presented as the means ± S.E., n = 3. B, quantification of 6-4 PPs obtained from immunofluorescent images. UV only samples (open triangles) and DPQ+UV samples (closed circles). Fluorescence intensity was normalized to untreated (NT) sample. The data are presented as the means ± S.E., n = 4. C, quantification of CPDs obtained from immunofluorescent images. Fluorescence intensity of raw data is shown. The data are presented as the means ± S.E., n = 3. *, p < 0.05; **, p < 0.01; ***, p < 0.01.

FIGURE 3.

UVR-induced association between XPA and PARP-1. HaCaT cells were exposed to a single dose of ssUVR (3 kJ/m²) and collected at the indicated times postexposure. A, representative image of co-immunoprecipitation. XPA was immunoprecipitated (IP) from cells, and the membranes were subsequently immunoblotted (IB) for PAR. The membranes were stripped and immunoblotted for XPA, as confirmation for immunoprecipitation, n = 3. B, representative image of co-immunoprecipitation. XPA was immunoprecipitated from cells, and the membranes were subsequently immunoblotted for PARP-1. The membranes were stripped and immunoblotted for XPA, as confirmation for immunoprecipitation, n = 3. C, dual staining with antibodies against XPA (red) and PARP-1 (green) was performed to assess the amount of co-localization (merge, yellow). D, quantification of intensities from C. The percentage of co-localization was determined as stated under “Experimental Procedures.” The intensities were normalized to the untreated (NT) sample. The data are presented as the means ± S.E., n = 3. **, p < 0.01. Scale bar, 10 μm.

FIGURE 4.

UVR-induced association between XPA and chromatin bound PARP-1. HEK 293 cells were exposed to a single dose of ssUVR (3 kJ/m²) and collected at the indicated times postexposure. A modified chromatin immunoprecipitation method (ChIP-on-Western) was then performed. A, representative image of co-immunoprecipitation. PARP-1 was immunoprecipitated (IP) from cells, and the membranes were subsequently immunoblotted (IB) for XPA. The membranes were stripped and immunoblotted for PARP-1, as confirmation for immunoprecipitation. B, quantification of Western blot by densitometry. NT, untreated. The data are presented as the means ± S.E., n = 5. *, p < 0.05.

FIGURE 5.

Silencing PARP-1 protein leads to decreased association between PARP-1 and XPA. Co-immunoprecipitations were performed in HaCaT and PARP-1 HuSH cells. A, representative image of co-immunoprecipitations. PARP-1 was immunoprecipitated (IP) from cells, and the membranes were subsequently immunoblotted (IB) for PARP-1, PAR, and XPA. B, representative image of reciprocal co-immunoprecipitations. XPA was immunoprecipitated from cells, and the membranes were subsequently immunoblotted for PARP-1, PAR, and XPA.

FIGURE 6.

Inhibition of PARP-1 activity leads to decreased association between PARP-1 and XPA. HaCaT cells were pre-exposed to a PARP inhibitor, DPQ, 30 min prior to UVR exposure. A, cells were collected 30 min post-UVR. Representative Western blot obtained from modified chromatin immunoprecipitation method (ChIP-on-Western). PARP-1 was immunoprecipitated (IP) from chromatin complexes. The membranes were immunoblotted (IB) for XPA and subsequently immunoblotted for PARP-1 as confirmation for immunoprecipitation. B, quantification of Western blot by densitometry. The data are presented as the means ± S.E., n = 3. C, HaCaT cells were pre-exposed to a PARP inhibitor, DPQ, 30 min prior to UVR exposure and cells were fixed 1 h post-UVR. Dual staining with antibodies against XPA (red) and PARP-1 (green) was performed to assess the amount of co-localization (merge, yellow). D, graph representing co-localization between XPA and PARP-1. The percentage of co-localization was determined using Pearson's correlation coefficient. The data are presented as the means ± S.E., n = 3. E, purified PARP-1 and His-XPA were incubated alone (lane 7), with activated PARP-1 (addition of activated DNA and NAD⁺, lane 8), or in the presence of the PARP inhibitor AG-014699 (lane 9). Following the various treatments, His-XPA was pulled down (IP) from each sample using cobalt-conjugated magnetic beads. Following pulldown, the membranes were subsequently immunoblotted (IB) for PARP-1, XPA, and PAR, NT=untreated. n = 3. *, p < 0.05; **, p < 0.01. Scale bar, 10 μm.

FIGURE 7.

Inhibition of PARP activity affects XPA function. HEK 293 cells were pre-exposed to a PARP inhibitor, DPQ, 30 min prior to UVR exposure and collected 5 min postexposure. A, representative Western blot obtained from modified chromatin immunoprecipitation (ChIP-on-Western). XPA was immunoprecipitated (IP) from samples, and its ability to bind chromatin was assessed by subsequent immunoblotting (IB). B, quantification by densitometry from ChIP-on-Westerns. XPA intensity was normalized to NT. The data are presented as the means ± S.E., n = 4.; **, p < 0.01.