PARP Inhibition Impairs Recruitment and Mobility of XPA, XPC and p53

PARP-1 influences various proteins by divergent modes. Covalent and non-covalent modification can control enzymatic activity, protein function, protein interactions, protein compartmentalisation, and finally its mobility and localization. In DNA single-strand break repair one of PARP-1’s most important tasks is the fast and efficient recruitment of the scaffold factor XRCC1 to sites of strand breaks. In Figure 4.24: XPA stimulates PARP-1 activity in-vitro. A. PARylation assay using recombinant PARP-1 and XPA in the presence of NAD⁺ and an activator oligonucleotide mimicking DNA strand breaks. Equal amounts of reaction mixtures were separated by SDS-PAGE, immobilized on a nitrocellulose membrane, and stained for PAR and XPA using specific antibodies. Histone H1 served as a positive control, BSA as a negative control. Displayed is one representative blot of five independent experiments. B. Semi-quantitative slot-blot PARylation assay using equal amounts of recombinant PARP-1 and increasing amounts of XPA, H1 and BSA. Top. Representative membrane stained for PAR using the mAB 10H. Bottom. Densitometric evaluation of three independent experiments, each performed in technical triplicates. Data represent means ± SEM. Statistical analysis was performed using One-Way ANOVA testing followed by Dunnett’s multiple comparison test. * p<0.05, *** p<0.001. Adapted from ⁴.

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Figure 4.25 it was investigated if a potential auxiliary influence of PARP-1 activity on NER was influencing the recruitment kinetics of NER-associated proteins to sites of DNA lesions. U2OS cells were transfected with plasmids coding for eGFP labelled DNA repair proteins. eGFP positive cell nuclei

Figure 4.25: PARP inhibition impairs recruitment of XPA-eGFP to sites of laser-induced DNA damage. Nuclei of U2OS cells were irradiated with a pulsed femtosecond laser as described in the Materials and Methods section.

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were irradiated with a pulsed femtosecond laser at a wavelength of 775 nm. This procedure results in multiphoton absorption in the confocal volume, mimicking light of a shorter wavelength (254 nm). This UV-C like energy absorption induces defined DNA damages like the typical NER photolesions CPDs and 6-4PPs ^545,547. Protein recruitment was followed over time using a laser scanning microscope.

As a positive control, recruitment of XRCC1-eGFP was monitored (Figure 4.25C & D). Laser irradiation caused a quick and strong increase of XRCC1-eGFP at the sites of the damage. During the first 40-50 s a steady slope of GFP signal could be detected after which a plateau was reached until the end of measurement at 75 s. This process of XRCC1-eGFP recruitment was so efficient, that the surrounding, undamaged nucleus was depleted of XRCC1-eGFP (compare fluorescence intensity remote of the damaged area in Figure 4.25C). Further, the recruitment of this protein was completely PAR-dependent. When the cells were preincubated with the PARP inhibitor ABT888, XRCC1-eGFP recruitment was abolished. Figure 4.25A & B shows the recruitment of PARP-1 itself and the formation of PAR at the sites of laser-induced DNA damage, which forms the basis of a PAR-dependent protein recruitment.

Figure 4.25E shows that XPA-eGFP is as well accumulating at sites of laser-induced DNA damage.

The depicted recruitment is slower and weaker as seen for XRCC1-eGFP, but unlike that protein, the XPA-eGFP signal intensity did not reach a plateau early on, but steadily increased over the full period of measurement (15 min). This possibly reflects the generally slower process of NER compared to the very fast single-strand break repair. ABT888 treatment significantly delayed the recruitment of XPA-eGFP (Figure 4.25F). This especially holds true for the very early phase of XPA-XPA-eGFP recruitment (first 20-40 s) in which PARP inhibited cells clearly showed slower XPA-eGFP kinetics. At later time points, PARP inhibition seemed to be less important for further XPA accumulation and the amount of recruited XPA-eGFP converged. 6-7 min after damage induction no differences between untreated and PARP inhibited cells could be observed.

PARP inhibition completely abolished the recruitment of XRCC1-eGFP, but not of XPA-eGFP. The observation, that XPA-eGFP still recruits when ABT888 was administered –even if slower as compared to cells with active PARP-1- suggests two separate mechanisms of XPA-eGFP recruitment, a PAR-dependent as well as a PAR-inPAR-dependent. Further, the converging of the recruitment slopes after 7 minutes indicates two relevant phases. While during the initial phase the PAR-dependent recruitment

A. Immunofluorescence microscopic analysis of XPA-eGFP transfected cells, demonstrating the local formation of poly(ADP-ribose) at ~1 min post irradiation. Cells were fixed with PFA and PAR was detected using the mAB 10H. B. Immunofluorescence microscopic analysis, demonstrating the recruitment of PARP-1 to sites of laser-induced DNA damage at ~4 min post irradiation. Cells were fixed with PFA and PARP-1 was visualized using the mAB FI23.C-D. Accumulation of XRCC1-eGFP at sites of laser irradiation and its inhibition by PARP inhibitor treatment (ABT888). C. Snapshot of cell nuclei before irradiation and 39 s post irradiation in the absence or presence of 10 µM ABT888. D. Recruitment kinetics of XRCC1-eGFP to sites of laser-induced DNA damage, acquired for 75 s in the absence or presence of the 10 µM ABT888. ABT888 completely blocked the recruitment of XRCC1 to sites of laser-induced damage. Data represent means ± SEM of 6 cells per condition. E-G. Accumulation of XPA-eGFP at sites of laser irradiation in the absence or presence of ABT888.

E. Time series of XPA-eGFP accumulation at sites of irradiation before irradiation (B.i.) and at different time points post irradiation as indicated (without PARP inhibition). F. Short-term recruitment kinetics of XPA-eGFP were acquired in the absence or presence of 10 µM ABT888. G. Long-term recruitment studies of XPA-eGFP.

Data represent means ± SEM of ≥10 cells analysed. Statistical evaluation was performed using Two-Way ANOVA analysis followed by Sidak’s post-test. * p<0.05, ** p<0.01, *** p<0.001. Scale bar indicates 10 µm.

Adapted from ⁴.

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seems to play a significant role, at later time points PARylation-dependent recruitment is either no longer taking place, or is completely covered by the slower PAR-independent XPA-eGFP accumulation.

4.4.4.2 Regulation of XPC and p53 Recruitment to Sites of Laser-Induced DNA Damage In the NER pathway, the protein XPC serves as an early factor of damage recognition and subsequent local helix unwinding. It is among the first proteins binding to sites of helix distorting DNA lesions. As described for XPA-eGFP, the PARP-dependent recruitment kinetics of XPC-eGFP were analysed using the same experimental settings described in chapter 4.4.4.1. Femtosecond laser irradiation at a wavelength of 775 nm resulted in absorbed energy levels of ~254 nm, causing CPD and 6-4PP DNA lesions. Protein recruitment kinetics were then followed with confocal live cell imaging (Figure 4.26A

& B).

Figure 4.26: PARP inhibition delays the recruitment of XPC-eGFP and p53-eGFP to sites of laser-induced DNA damage. Nuclei of U2OS cells were irradiated with a pulsed femtosecond laser as described in the Materials and Methods section. A. Recruitment kinetics of XPC-eGFP to sites of laser-induced DNA damage, acquired for 600 s in the absence or presence of 10 µM ABT888. ABT888 significantly impaired the recruitment of XPC-eGFP to sites of laser-induced damage. Data represent means ± SEM of 16 cells per condition. Statistical evaluation was performed using Two-Way ANOVA analysis followed by Sidak’s post-test. B. Representative images of pEGFP::XPC-eGFP transfected cells before and after laser irradiation. C. Recruitment kinetics of p53-eGFP to sites of laser-induced DNA damage, acquired for 300 s in the absence or presence of 10 µM ABT888. ABT888 significantly impaired the recruitment of p53-eGFP to sites of laser-induced damage. Data represent means ± SEM of 5 cells per condition. D. Representative images of pEGFP::p53-eGFP transfected cells before and after laser irradiation. Statistical evaluation was performed using Two-Way ANOVA analysis followed by Sidak’s post-test for the data obtained in the first 170 s. **** p<0.0001.

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XPC-eGFP recruitment was in general more prominent than seen for XPA. Similar to XRCC1-eGFP dynamics, XPC-eGFP recruitment reached its maximum 2-3 min after irradiation. After 10 min protein levels at the sites of DNA damage already seemed to decrease again, although they still retained at high levels. PARP inhibition caused a similar, but even stronger effect as seen for XPA-eGFP. Early recruitment of XPC-eGFP was significantly delayed (first 1-2 min), but accumulation steadily continued till the end of measurement. After 10 minutes the slope was still increasing and had not reached, or converged with XPC-eGFP levels of untreated cells. Further, in PARylation deficient cells XPC-eGFP did not show any signs of dissociation from the sites of DNA damage.

The transcription factor p53 is not directly involved in DNA repair, but has been reported to facilitate NER by transcriptional control of the GG-NER factors DDB2 and XPC ³³³. Here, the same experimental setup was utilized to analyse the recruitment kinetics of the p53 (Figure 4.26C & D). A mild, but clear recruitment of p53-eGFP to sites of laser-induced DNA damage was observable (5-10 %). Kinetics were comparable to the recruitment of XPA-eGFP, showing a fast immediate recruitment, followed by a steady, but less steep rise of protein levels. Different from XPA-eGFP, p53-eGFP levels seemed to reach a plateau within the time frame analysed. Further, PARP inhibition significantly delayed recruitment of p53-eGFP, especially early after damage induction. About 6 min after irradiation the amount p53-eGFP at the damage site in PARP inhibited cells was comparable with the levels in uninhibited cells.

4.4.5 Altered Mobility of a PAR-Binding Deficient XPA Variant