Reciprocal Regulation of XPA-PARP-1 Protein Functions

Two different approaches were performed to analyse the influence of PARP-1 activity on NER efficacy (Figure 4.21). On the one hand, cells were UV-C irradiated and the repair kinetics of photolesions in cells was followed by the use of CPD specific antibodies. Herein, PARP inhibited cells displayed a significantly reduced DNA repair rate of CPDs within the first 24 h after damage induction. In a second approach, the general NER capacity of an UV-C irradiated reporter plasmid was determined by an HCRA. Here, cells with active PARP-1 protein showed higher capability to restore the reporter gene expression compared to PARP inhibited cells (~10%). These observations are perfectly in line with very recent reports, showing impaired repair of UV-induced photolesions in cells with deficient PARP activity ^483,485. Several mechanistic studies followed with a focus on a role of PARylation in initial lesion recognition by DDB2/XPC in the GG-NER and by CSB in TC-NER 483,485,553. Although being the earliest identified NER factor to interact with PAR, little attention has been attributed to the rate-limiting factor XPA.

Protein-PAR interaction can have a plethora of possible consequences. Defining cellular localization, alteration of protein stability, inducing or interfering with enzymatic activities and regulating protein-protein interactions of target protein-proteins and downstream factors are some of the major implications ⁶⁰. A

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PAR chain is a negatively charged molecule. Thus, it is easy to imagine that the introduction of these negative charges by PAR-binding might interfere with XPA’s interaction with other negatively charged molecules such as DNA. Furthermore, the close proximity of XPA’s DNA-binding site to its PBM, as well as the necessity for long polymer makes it conceivable that additionally to a putative electrostatic repulsion also steric hindrance might come into play. Finally, very recent studies suggested that the region covering the PBM, although not being essential, might contribute to XPA’s full DNA-binding capability ^3,500.

Considering XPA’s strong preference for long and branched polymer, electrophoretic mobility shift assays with size-fractionated PAR were conducted to analyze the functional consequences of this preference on XPA’s DNA-binding affinity. As a binding substrate biotin-labeled duplex DNA, carrying a loop structure, was used. When performing EMSAs with recombinant XPA and this substrate, a mobility shift could be observed, suggesting XPA binding to the DNA. Co-incubation of XPA with DNA and long polymer (51-55mer) interfered significantly with the XPA-DNA interaction.

Increasing concentrations of PAR chains resulted in a backshift to protein-unbound DNA, implicating an inhibitory influence of PAR on XPA’s DNA-binding capabilities (Figure 4.23). Repeating these experiments with short chained polymer (16-20mer) on the other hand revealed, that identical concentrations of PAR chains had no influence on the XPA-PAR interaction. This is in line with the previous observation of size-specific PAR-binding, but provides for the first time a functional consequence of this preference.

Up to now, it is not completely understood how the numerous effects and influences of PARP-1 and PAR are controlled and specificity is guaranteed. The mechanisms behind the specific impact on the ever increasing numbers of PAR-interacting proteins remains mainly elusive. Besides crosstalk between different kinds of PTM, it could be imagined that a size-selective interaction provides another level of control. In the case of NER, it could be assumed that the early and transient induction of PARylation induced by DDB2-interaction, might not be strong enough for a lasting influence on XPA’s DNA-binding affinity but might still be strong enough for its role in the initiation of the DNA damage recognition by DDB2 and XPC. On the other hand, a second peak of PARylation, due to the occurrence of strand incisions, persisting DNA lesions or other forms of PARP stimulation might produce PAR chains of sufficient length to mediate XPA’s dissociation from the DNA lesion site.

XPA serves as a scaffold factor in the NER pathway. Together with TFIIH it verifies the DNA lesion, orchestrates the assembly of the repair machinery and controls correct placement of the endonucleases.

To fulfill these tasks, XPA interacts with nearly all central NER factors. Binding to PAR might herein play a regulatory role. A significant portion of XPA is intrinsic disordered 528,554,555, which most likely allows the interaction with such a wide array of proteins. Both of XPA’s PAR-binding sites are located within such disordered regions and it is conceivable that upon PAR-binding conformational reorganizations might take place, which in turn could influence XPA’s protein-protein interactions.

Thus, it is of interest to further characterize the XPA-PAR interaction in respect to XPA’s protein interactome. This might help to understand how the complex mechanisms of NER preincision complex assembly are organized and maintained.

During the repair of single-strand breaks one of the major implications of PARP-1 activity is the rapid recruitment of the scaffold factor XRCC1, essential for the assembly of the repair machinery and the subsequent processes ^370,556. XPA can be assigned comparable attributes as for XRCC1 in respect to the organisation of the NER pathway.

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To determine whether PARylation affects the spatio-temporal localisation of XPA in response to UV irradiation, live cell microscopy of cells transfected with a XPA-GFP expression constructs was conducted. Local DNA damage was induced by the use of a pulsed femtosecond laser, which induces among others, the typical UV-photolesions 6-4PP and CPDs in a defined local area. Recruitment kinetics to the sites of damage induction were then determined via laser scanning microscopy (Figure 4.25).

Compared to XRCC1, XPA recruitment is a slow and less intense process. At the sites of DNA damage, intensity of XRCC1-eGFP increased within seconds more than 8-fold, while the recruitment of XPA-eGFP only increased by about 15 % in a time scale of minutes. Further, a biphasic recruitment kinetic for XPA could be observed, an early, very rapid increase of XPA at the site of damage was followed by a slower, steady accumulation of the protein. This is in line with the literature, reporting a steady increase of XPA at the sites of DNA damage within the first 60 minutes, and a slow dissociation taking hours ^468,557. Inhibition of PARP-1 activity completely abolished XRCC1 recruitment to lesion sites, proofing the dependence of XRCC1 on PARylation. For XPA also a significant reduction of recruitment kinetics could be observed upon PARP inhibition. This holds especially true for the early phase of XPA recruitment (~6 min). Thereafter, comparable intensities of XPA-eGFP at irradiation sites were detected in cells with active and inactive PARP. This finding implicates a two-phased recruitment of XPA to damage sites. Initial recruitment of XPA is actively facilitated by PARylation, but its relevance diminishes over time.

Of note, when repeating these experiments with XPC-eGFP, similar, but even enhanced effects were obtained (Figure 4.26A). XPC recruits to the lesion sites in a stronger manner than XPA, and PARP-1 inhibition provoked clearer and slightly longer lasting effects. XPC is modified by PARylation and its cellular mobility has been shown to be influenced by PARP-1 activity before ^98,485. Since XPC is the key damage recognition factor in the GG-NER sub-pathway, its presence at the lesion site likely precedes the presence of XPA. Thus, the faster and stronger recruitment of XPC compared to XPA is probably not surprising. At this point, it cannot be excluded that the delay of XPA recruitment upon PARP inhibition is not a consequence of the reduced damage recognition by XPC. To distinguish between a direct XPA-PAR recruitment and an altered PAR-XPC-XPA axis, a PAR-binding deficient XPA mutant would be advantageous.

Finally, the same experiments were repeated using a p53-eGFP expression construct (Figure 4.26B).

p53 has been shown to be regulated via PARylation by several means (see 1.2.4). But on the other hand, p53 is not supposed to be directly involved in DNA repair processes but to be a master regulator of transcriptional activation upon genotoxic stress 186,187,333,558. Therefore, it is somewhat surprising to see a weak, but clearly active recruitment of p53 to sites of laser-induced DNA lesions. Furthermore, PARP inhibition delayed this recruitment in a similar manner as seen for XPA. Although an unspecific recruitment of p53 due to its non-covalent interaction with PAR cannot be excluded at this point, it should again be stressed out that the manifold influences of PAR on the large body of cellular proteins is neatly controlled. Further, the presence of a PAR-binding module does not necessarily result in a PAR-dependent relocalisation, as seen for example for the DEK protein (A. Deutzmann, personal communication). It could be assumed, that recruitment of p53 is not related to a direct repair function at the lesion site. But this site is necessarily a region of active PARylation, thus the recruitment of p53 to sites of active PARylation might in turn facilitate its UV-irradiation-induced covalent modification, and subsequently altering of p53’s transcriptional activity.

Based on the peptide mutation studies two XPA-GFP constructs were generated with diminished PAR-binding of XPA’s C-terminal PBM (aa 210-237) (Figure 4.20A). Site-directed mutagenesis and cloning

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of these constructs were successfully conducted and in the course of this thesis one of the XPA mutants (XPAmut2; R227A, R228A, R231A) was tested in a pilot experiment. Here, cells were transfected with either XPAwt-eGFP or XPAmut2-eGFP expression constructs, FRAP analysis was performed and the mobility of proteins in unstressed cells were compared (Figure 4.28).

Photo-bleaching reduced the local fluorescence intensity by about 50 % and immediately thereafter fast recovery of XPA-fluorescence intensities was observed. Comparable time scales of recovery (within seconds) have previously been reported for other NER proteins ⁴⁸³. This outlook experiment was conducted in unstressed cells, therefore determining XPA’s general mobility. Interestingly, XPAmut2-GFP showed a weak, but significantly higher degree of mobility. Even in unstressed cells, there is a steady-state level of PAR formation, which might already be sufficient to significantly slow down mobility of XPAwt via non-covalent PAR-binding. On the other hand, in this study a second N-terminal PBM has been identified which still should contribute to XPA’s PAR-binding ability. The mutated PBM (aa 210-237) is located in close proximity to the MBD of XPA and furthermore even in a region which was previously suggested to contribute to XPA’s full DNA-binding affinity. In this region several lysine residues have been identified, which were supposedly important for this contribution ³. Amino acid alterations in this area might thus decrease XPA’s DNA-binding affinity and the observed protein-mobility differences might be a result of decreased DNA-binding rather than decreased PAR-binding.

Anyhow, these lysine residues were not altered within the XPA-PBM mutant analysed in this study.

However, it cannot be excluded at this point, that the site-directed mutagenesis within XPA did not alter its DNA-binding affinity.

Thus, in follow-up experiments first the DNA-binding affinity of XPAwt and XPA-PBM_mut variants should be compared by using, XPA-DNA EMSAs or SPR analysis. Second, the newly identified N-terminal PBM should additionally or alternatively be mutated within full length XPA, to further decrease XPA’s PAR-binding affinity and possibly increase putative mobility differences. Here, it has to be taken not to disturb the NLS of XPA. Finally, the FRAP experiment should be repeated in a cellular context of increased genotoxic stress stimulating PARP-1 activity, such as UV-C irradiation.

Additionally, a PAR-binding deficient XPA mutant should be used to validate the results obtained so far by in-vitro as well as in-vivo assays, such as PAR influence on XPA’s DNA-binding and PAR-dependent recruitment of XPA to DNA-lesion sites.

The role of PARP-1 is probably best studied and understood in the repair of DNA single-strand breaks (Figure 1.4). Here, PARP-1 detects these strand discontinuities, binds via hydrophobic interaction to exposed bases, resulting in conformational alterations and induction of PARP-1 activity ^40,78. The trigger of PARP-1 activation in the NER pathway is less well understood. PARP-1 becomes activated at sites of UV-photolesions but the mechanisms behind this strand break-independent activation remains mainly elusive ^483,559. Meanwhile, several DNA lesion-independent modes of subtle PARP-1 stimulation have been described, as PTM, SIRT6-mediated ‘kickstart’ via MARylation, and direct protein-protein interaction ^69,71,74. Indeed, an interaction of PARP-1 with DDB2 was suggested to be the trigger of initial PARylation events in the NER pathway ⁴⁸³. However, experiments performed in this thesis revealed a second, or supportive mode of PARP-1 activation.

To test whether the presence of XPA directly influences PARP-1 activity two different in-vitro approaches were conducted (Figure 4.24). In both assays, recombinant PARP-1 was mixed with sufficient amounts of NAD⁺, a PARylation stimulating DSB-mimic duplex oligonucleotide and either recombinant XPA or control proteins. For the first approach, the reaction was allowed to run for a comparative long period of time (15 min). As mentioned earlier, the first target of PARylation is PARP-1 itself and upon sufficient autoPARylation, PARP-1 activity is diminishes as part of an

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autoregulatory mechanism. Therefore, after 15 minutes of reaction with excess NAD⁺, PARP-1 activity should be saturated and the amount of detected polymer corresponds to the maximal amount of possible PAR formation under that condition.

When this assay was conducted in the presence of histone H1, PAR synthesis was clearly stimulated as a response to the presence of an additional PARylation target. Similar to H1, recombinant XPA stimulated PARP-1 significantly. Even in the absence of the activating oligo DNA, XPA clearly stimulated the otherwise very basal PARP-1 activity. This suggests a direct protein-protein mediated PARP-1 stimulation.

This assumption was further supported by the second in-vitro PARylation approach. This time the reaction was stopped after 30 seconds, when PARylation is still situated within the dynamic phase. Thus detected PAR corresponds to the speed of the initial PARP-1 activation. Here, the presence of H1 had only little influence on the degree of PAR formation, suggesting a minor role of the presence of additional PARylation targets during the initial phase, which becomes only more relevant in ongoing reaction by delaying the PAR saturation. XPA on the other hand again induced a strong and concentration-dependent PARP-1 stimulation.

Figure 5.1: Simplified model of the role of PARP-1 within the NER process. Helix-distorting photolesions, such as CPDs, are detected and bound by the UV-DDB complex. DDB2 in turn recruits and stimulates PARP-1 at the site of DNA lesion, stabilizing DDB2 itself and facilitating the recruitement of the downstream NER machinery. XPA is recruited, binds to the lesion site and further stimualates PARP-1 activity, by which its own fuctionality is regulated.

Ultimatly, this results in inactivation and dissociation of PARP-1 of the repair complex. Adapted from ⁴.

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Thus, the XPA-PARP-1 interaction might contribute to the NER stimulated PARylation activity.

Situated downstream of DDB2 (probably after its degradation), XPA might take over the task of PARP-1 stimulation even forgoing strand incision and thus further coordinating the PAR-dependent NER mechanisms, while simultaneously regulating its own PAR-dependent functions.

From the biochemical and cell biology-based experiments performed within this thesis a novel conceivable sequence of XPA- and PARP-1 dependent events in the NER of UV-photolesions can be proposed (Figure 5.1):

As a prime event in the GG-NER, the UV-DDB complex detects and binds poorly helix-distorting DNA