Biochemical Analysis

5.1.1 Biochemical Prerequisites

Particularly for the first part of this thesis, several biochemical prerequisites had to be established.

Recombinant PARP-1 protein was overexpressed in Sf9 cells and purified by means of salting out, size exclusion and affinity chromatography (Figure 1.1). This purification procedure resulted in sufficient amounts of active protein of rather good purity. Depending on the degree of purity, recombinant PARP-1 was either used in biochemical assays or for medium-scale in-vitro PARylation. Herein synthesized polymer was used as a mixture of PAR of varying length, or for more specific purposes was further processed and size fractionated by using anion exchange HPLC (Figure 4.13).

Strategies of High Five-overexpressed His-XPA purification were successfully transferred to a FPLC-based system. Purification was performed in two subsequent affinity chromatography steps, using Ni Sepharose HP and Heparin HP containing columns. Resulting protein yields were good and depending on the fraction of excellent purity (Figure 4.6 and Figure 4.7).

Finally, an attempt was made of cloning constructs to overexpress and purify five His-XPA fragments, covering all of its essential domains. The lack of a functional DNA-binding site in these fragments made it necessary to modify purification strategies. Replacing Heparin HP affinity purification with size exclusion chromatography generated XPA fragments of excellent purity and sufficient yield. In the course of this study, it was possible to accomplish the purification of XPA-F1, XPA-F2 and XPA-F3 (covering the domains from aa 1-210), with XPA-F3 only processed in a one-step IMAC purification (Figure 4.8-Figure 4.10). Overexpression of XPA-F4, which primarily contains XPA’s PBM (aa 213-237), turned out to be toxic for High Five cells. Cytotoxicity caused by overexpression of recombinant proteins is a frequently observed feature. Alteration of expression conditions (MOI, temperature, expression period) had no influence on the degree of toxicity. Switching to another expression system (e.g. yeast, E. coli) might help to overcome this obstacle. The generation of the XPA-F5 overexpression-construct was successfully accomplished, but protein overexpression in High Five cells and subsequent purification could not be completed in the time frame of this thesis.

5.1.2 Biochemical Characterization of the PAR-XPA Interaction

The regulatory influence of PARP-1 is archived by two modes, covalent PARylation of target proteins or non-covalent interaction with the polymer, using one of the PAR-binding modules. The most abundant of these modules is the PBM, a small patch of alternating hydrophobic and basic amino acids found in dozens of proteins mainly involved in DNA repair and cell cycle control. Although being the first module identified to non-covalently bind PAR, this motif remains rather poorly characterized. XPA was among the first proteins in which this motif was described. The PBM of XPA is located in the C-terminal half of the protein at the interface between the minimal DNA-binding domain and the TFIIH interaction site (aa 213-237) ¹⁴⁷.

In this thesis, the non-covalent interaction between XPA and PAR polymer was verified by far-western blotting (PAR-Overlay Assay), successfully confirming XPA’s PAR-binding ability as reported earlier (Figure 4.14C) ¹⁴⁷. To address the functional consequences of this PAR-XPA interaction, a closer look at the biochemical properties of XPA’s PBM was taken.

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An in-silico analysis approach was performed by aligning XPA’s amino acid sequence of different species, ranging from human to worm. Overall, varying degrees of conservation were found. While the N- and C-terminal portion of the protein are less conserved among species, the MBD showed a striking amount of sequence homology (data not shown). Of note, the stretch of highest conservation extends beyond the MBD and covers XPA’s PBM, emphasizing the importance of this region (Figure 4.15A).

Within this PBM four amino acids, three basic and one hydrophobic, are conserved across all species.

This is of special interest since such amino acid residues were previously suggested to be most relevant for a functional PBM in other proteins ^147,264.

For the analysis of the interplay between XPA and PAR in a cellular background, it is a suitable approach to use PARP inhibitors or PARP-1 knockout cell lines. But using such systems is accompanied with some downsides, such as simultaneously affecting all of the manifold PARP-1-dependent pathways and thus problematic assignment of the seen effects. Further, upon knock-out of PARP-1, several functions can be compensated to some degree by other members of the PARP family, as suggested earlier ⁵⁴⁸. On the other hand, current PARP inhibitors always target more than one PARP family member, thus possibly influencing several unrelated functions ³⁶. Disturbing the NAD⁺-SIRT1 axis as well as potential PARP trapping and the resulting genotoxic stress can cause additional side effects ^104,215. The use of mutant protein variants, no longer able to interact with PAR, provides a more favourable system for a specific analysis of the regulatory role of a protein’s PBM.

To generate such PAR-binding deficient XPA variants, it is essential to determine which of the amino acids within the PBM are most relevant for the PAR-interaction, in order to minimize the extent of mutation. Recent studies suggested a higher relevance of basic amino acids within the motif ^155,264. Thus, these were exchanged to alanine in order to interfere with PAR-binding (PBM1:K217A, K218A, K221A, K222A and PBM2: R227A, R228A, R231A). Short peptides of XPA’s PAR-binding region (25 aa), containing the appropriate mutations, were synthesized and their ability to non-covalently interact with PAR was investigated in a PAR-overlay assay (Figure 4.15C). A peptide covering XPA’s wild type PBM served as control. The PAR-overlay assay revealed that this short amino acid sequence is sufficient for XPA’s PAR-binding ability (PBM wt) and the presence of basic amino acids were identified to be essential for the interaction. Both sets of PBM mutations completely abolished PAR-binding.

Besides the identification of the PBM by Pleschke et al., the interaction between XPA and PAR is only poorly characterized ¹⁴⁷. A subsequent study by our group performed PAR-EMSAs and SPR analyses to define the affinity coefficient of this interaction ¹⁵². Interestingly, a positive correlation between PAR chain length and binding strength to XPA was found. A similar observation could be made for the DEK protein, but is not existing in many other PBM containing proteins ¹⁵³. The mechanism behind this size-selective interaction remains elusive so far. It is imaginable that specific structural features, defined by the PBM, provide for the size selectiveness. PAR chain length correlates with the chance of branching events. Thus, one could hypothesize that size-selective PAR-binding is mediated by an increased affinity to PAR-branching points over linear polymer. The structural details of the PBM-PAR interaction are largely unknown, but it was suggested that that the specific PBM consensus sequence is of less importance compared to general structural features ⁶⁰. Indeed, XPA’s PBM shows only a weak resemblance to any proposed consensus sequence (two mismatches within the eight core PBM amino acids). These differences might alter the general structure towards an enhanced preference for branched PAR. On the other hand, it is possible that the PBM itself is only a weak PAR binder, but general electrostatic interactions between the protein surface close-by the PBM and PAR could contribute to the binding. This assumption is probably supported by the observation that XPA’s PBM is poorly conserved, but indeed four partial overlapping core-motifs could be located in this region (data not

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shown). In this case, polymer of a certain length would be necessary for a tight interaction. Related to this, it could be supposed that a second PBM is essential for efficient XPA-PAR binding and again, PAR of a certain length would be a prerequisite for an interaction with both PBMs.

In order to analyse if additional PBMs are present within XPA, possibly contributing to PAR size-selective binding, purified fragments of XPA were analysed for PAR-binding abilities via PAR-overlay assays (Figure 4.16 - Figure 4.19). Three XPA fragments were tested: F1 containing the N-terminal portion of XPA (aa 1-97), F3 resembling XPA’s MBD (aa 98-211) and F2, overlapping with these two domains (aa 60-140). Neither for XPA-F2 nor for XPA-F3 non-covalent interaction with the polymer was detectable. Interestingly, XPA-F1 proofed to be a strong PAR-binder. Since XPA-F2 revealed no affinity for PAR, the binding site within F1 could be narrowed to the very N-terminal region of XPA (aa 1-60).

Related to this finding, in-silico analysis was performed in order to search for additional, putative PBMs in XPA’s amino acid sequence. Several possible PBMs could be identified, although they showed weak consent (two mismatches). Similar to the previously identified PBM, these regions [aa 23-49 and aa 148-175 (data not shown)] were composed of several overlapping PBM core motifs. Peptides of both regions were synthesized and tested for their affinity to bind the PAR polymer. In line with the results obtained by the XPA fragments, the latter peptide (aa 148-175 residing in XPA-F3) did not bind PAR (data not shown). On the other hand, peptides of the two N-terminal core-motifs revealed a strong affinity for PAR (Figure 4.20B). Of note, these PBMs overlap with several other important interaction sites. Via its N-terminal part, XPA interacts with CEP164, a protein involved in G2/M checkpoint signalling, relevant for nuclear division and key factor in response to replication stress and DNA damage by UV light and ATR signalling ⁵⁴⁹. Further, this region coincides with the RPA32-binding site and XPA’s NLS.

Based on the identification of an additional PBM within XPA, it is tempting to speculate on possible consequences of XPA’s PAR-binding. First, as mentioned above, this newly identified PBM could contribute to XPA’s size-selective binding to polymer. Here, it should be mentioned that this PBM has been previously identified in-silico, but was rejected after in-vitro testing ¹⁴⁷. However, in their study size-fractionated PAR of short chain length (15/16mer) was used. Assuming that the size-selectiveness for PAR chains resides within the PBM itself, this could explain why it was tested negative in that study.

Further analysis using polymer of different composition and length with this PBM is needed to shed light hereon. Anyhow, it is conceivable that this newly identified PBM contributes to XPA’s enhanced affinity for long and branched polymer.

Furthermore, it is tempting to speculate that the overlap of the newly identified PBM with the NLS plays a regulative role in XPA’s cellular localization. The NER pathway detects a wide range of DNA lesions, but several factors, including XPA, have also been identified to show some affinity for undamaged DNA (DNA probing) ⁴⁶⁸. This holds some threat for unspecific, damage-independent DNA processing. Different modes have been suggested to guarantee NER specificity. On the one hand, the sequential assembly of the NER machinery is accompanied by several detection and verification steps (XPC, TFIIH and XPA). Other mechanisms include the general limitation of NER initiation factors, which can be up-regulated in response to UV-irradiation (DDB2 and XPC) ^550,551. Although such transcriptional regulation was not reported for XPA, it has been shown that a portion of the protein can be found in the cytoplasm, which relocates rapidly to the nucleus upon DNA damage ⁵²³. Further, UV-irradiation provokes two rounds of PARylation: an early DDB2-dependent and later PARylation peak, which is probably due to emerging of NER-induced DNA strand breaks ^483,488. PARylation upon UV-irradiation might thus influence XPA levels in the nucleus, as it was reported for other proteins (p53,

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NF-κB) ^188,232. Here, both would be conceivable, an increased nuclear import, probably due to facilitating protein-protein interactions essential for nuclear shuttling (XAB1, importin-4α) ^523,552, as well as an increased export by PAR-dependent masking of the NLS, to limit XPA abundance after lesion repair (second PARylation peak).

Similar assumptions are conceivable for XPA’s interaction with RPA. XPA was shown bind DNA only with moderate affinity, which significantly increases upon interaction with RPA ⁵⁰⁹. Here, PARylation might be involved in mediating this protein-protein interaction and thus XPA’s affinity for the lesion site.

Simultaneously to the search for additional PBMs in XPA, three promising in-silico identified PBMs of HERC2 were analysed (Figure 4.20B). All three peptides were found positive, identifying HERC2 as a novel PAR-binder. This is of interest, since this protein is a central regulator of XPA’s turnover.

Via its E3 ubiquitin ligase activity, it marks XPA for degradation. XPA is a comparably short lived protein and protein levels oscillate according to the circadian clock ⁵²⁰. Further, upon UV-irradiation degradation of XPA is reduced, probably due to destabilization of the XPA-HERC2 interaction as a result of deacetylation of K63 and K67 by SIRT1 ⁵¹⁵. An additional layer of protein level control could be facilitated by rapid PAR-dependent regulation of HERC2 activity. Here, it can be assumed, that UV-irradiation stimulates PARP-1, which in turn controls HERC2 activity via non-covalent PAR interaction with its PBMs. This might contribute to the orchestration of protein levels of DNA repair factors, such as XPA, in response to DNA damage. Crosstalk between PARylation and ubiquitination has been shown in several pathways. The majority of the WWE domain containing proteins as well as one PBZ containing protein are E3 ubiquitin ligases ⁶⁰. Further, PARylation of axin was identified to be a prerequisite for ubiquitin mediated degradation (Wnt/β-catenin signalling). On the other hand, PARylation interfered with ubiquitination of the NER factor DDB2, thus stabilizing protein levels ^60,483. Of note, HERC2 is an important ubiquitin ligase involved in mediating degradation of several DNA repair associated proteins (e.g. BRCA1) ⁵²¹. Anyhow, the identification of HERC2 as a PAR-binder marks the first ubiquitin ligase interacting with PAR via the PBM. Analysis of the functional consequences of a HERC2-PAR interaction might help to understand the dynamic processes occurring at lesion sites in the context of UV-irradiation, or on a broader perspective, in the general regulation of DNA repair factors in response to genotoxic stress-induced PARylation.