XPA Non-Covalently Interacts with PAR

4.3.1.1 Scheme and Model of XPA

XPA is embedded at the very centre of the nucleotide excision repair pathway. It serves as an organizer and scaffold factor for the many proteins involved in lesion recognition and excision. Thus it is not very surprising, that the interaction partners of XPA are manifold. In Figure 4.14A a scheme of the predicted secondary structure of XPA, with some of its major interaction partners, is depicted. XPA carries an N-terminal nuclear localization sequence (aa 26-47), a central minimal DNA-binding domain (MBD, aa 98-219), including a zinc finger (aa 105-129) and a C-terminal TFIIH-binding domain. Partially overlapping with the MBD the PBM (aa 213-237) can be found. XPA is known to be acetylated (K63 and K67), phosphorylated (S196) and ubiquitinated. Figure 4.14B shows the NMR-model (‘1XPA’) of

Figure 4.13: Modified silver gel showing in-vitro synthesized PAR.

PAR was synthesised using recombinant PARP-1 and subject to a 20 % TBE-PAGE and subsequent silver staining. (Right). 1 or 5 nmol of unfractionated PAR was applied.

(Above). PAR was size-fractionated using anion exchange HPLC.

Contributions by [B].

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a partial sequence of XPA. A PAR-overlay assay was performed, depicted in Figure 4.14C, to confirm that XPA binds PAR with a high affinity in a non-covalent manner. Full-length recombinant proteins were separated by SDS-PAGE and subsequently analysed by far-western blotting. The membrane was incubated overnight with in-vitro synthesized PAR. Unspecific-bound polymer was removed by high salt washing and non-covalently bound PAR immunochemically detected. The PAR-overlay in Figure 4.14 demonstrates XPA binding PAR with a comparable affinity as the positive control histone H1.

Multiple bands reflect distinct confirmations of XPA ^489-491.

4.3.1.2 XPA’s PBM is Highly Conserved among Species and Basic Amino Acids Within are Essential for PAR-Binding

Having confirmed XPA’s ability to bind PAR non-covalently, a closer look was taken on XPA’s reported PBM (aa 213-237). Pleschke et al. described the PAR-binding motif as an alternating pattern of basic and hydrophobic amino acids ¹⁴⁷. XPA’s PBM is only weakly consistent in this respect.

Aligning it with the general PBM pattern reveals at least two mismatched amino acids, but on the other hand, several partially overlapping core motifs could be identified within this small area of the protein.

A sequence alignment of the human XPA-PBM and of the homologous region from different species with known or predicted PARylation activity was performed (Figure 4.15A). Although not being part Figure 4.14: XPA binds PAR in a non-covalent manner. A. Functional domains of XPA including predicted secondary structure elements. The PAR-binding motif (PBM) is indicated in green, the Zinc finger domain (Zn finger) in red, the DNA-binding domain in black, the nuclear localization signal (NLS) in blue, and protein-protein interaction domains in orange. B. NMR model of the minimal DNA-binding fragment of human XPA (aa 98-210) based on PDB file ‘1XPA’ ². The Zn finger domain is highlighted in red, the RPA70- and DDB2-interaction domains in orange. The PBM is located next to the C-terminal end of the structure shown, and was not part of the analysed fragment. C. PAR overlay blot demonstrating non-covalent XPA-PAR interaction. Proteins (in amounts indicated) were separated by SDS-PAGE, immobilized on a nitrocellulose membrane, and incubated without or with 0.2 µM PAR. After high salt washing of membranes, protein-bound PAR was detected using the anti-PAR mAB 10H. PAR binding to histone H1 served as a positive control, absence of PAR binding to bovine serum albumin (BSA) as negative control. Adapted from ⁴.

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of XPA’s highly conserved MBD a strikingly high level of homology in the PBM could be observed between species, highlighting the importance of this region on XPA’s functionality. Of note, three basic and one hydrophobic amino acids were identified to be conserved in all species tested. This is of special interest, since these two classes of amino acids are supposed to be most relevant for the non-covalent PAR-protein interaction ¹⁴⁷. To determine in detail which of the amino acids are essential for XPA’s PAR-binding ability, and to provide a basis for the generation of a XPA PAR-binding mutant, a peptide approach was conducted. Two peptides of XPA’s PBM with exchanged basic amino acids to alanines, were custom synthesized and their PAR-binding ability was compared to the XPA wt peptide (Figure 4.15B & C).

Figure 4.15: The XPA-PBM is highly conserved among species. A. Sequence alignment of the PBM of XPA from various species revealed a high degree of homology. Amino acids with similar biochemical properties are indicated by the same shades of grey. B. Two mutant versions of XPA’s PBM were generated, with basic amino acids exchanged to alanine. C. Two XPA PBM mutants (aa 213-237) were tested for their PAR-binding affinity after sequence alteration. Both mutants were shown to lack affinity for PAR. Adapted from ³.

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The mutant versions of XPA’s PBM were slot-blotted on a nitrocellulose membrane and incubated overnight in PAR. After stringent washing in high-salt buffer, non-covalently bound PAR was detected (Figure 4.15C). Both mutant peptides lacked PAR-binding, identifying the basic amino acids to be essential for XPA’s interaction with PAR.

4.3.1.3 The XPA’s N-terminal Fragment XPA-F1 Interacts with PAR

As mentioned before, XPA’s PBM is only weakly consistent with the general PBM pattern proposed by Pleschke and colleagues. Applying an in-silico approach several other putative PBMs of such or higher consistency could be identified in different domains of XPA. To evaluate the existence of additional functional PBMs within XPA, the XPA fragments purified earlier were tested for their PAR-binding abilities.

Therefore, 5 pmol of the positive control H1, 50 pmol of the negative control BSA and 5, 50 or 150 pmol of each XPA fragment were size separated by SDS-PAGE and subject to far-western PAR overlay assay. Equal loading of proteins was verified by subsequent staining for the recombinant proteins with an anti-His-tag antibody.

Figure 4.16 depicts a PAR overlay of the XPA fragment 1 (aa 1-97). Here, 10 pmol purified full-length XPA was included as well. The left hand panel shows the membrane incubated in PAR solution. A strong signal can be seen for the positive control H1, no signal was detectable for the negative control BSA. Interestingly, at 17.5 kDa a clear PAR signal was observed, which was dependent on the amount of applied recombinant protein. Further, full-length XPA also bound PAR, resulting in a signal comparable to the intensities ranging between the 5 and 50 pmol of F1.

Figure 4.16: PAR overlay blot with recombinant XPA-F1 fragment. Positive control histone H1, negative control BSA, full-length XPA and the N-terminal fragment of XPA (XPA-F1, aa 1-97) were size separated via SDS-PAGE and subsequently analysed for their non-covalent PAR-binding abilities by far-western blotting. After blotting, the membrane was cut in two identical parts. Membranes were incubated overnight in TBS-T containing 0.2 µM PAR (+PAR) or only in TBS-T (-PAR). Immunostaining of PAR indicated a XPA-F1-PAR interaction (Left).

Visualization of His-tag was used as a loading control (Right). One representative blot of two independent experiments is shown.

Figure 4.17: In-silico identified PBMs in XPA-F1. Three potential core PBMs can be found within XPA’s N-terminal region.

Inconsistencies to the proposed PBM are highlighted in red.

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The central panel visualizes the specificity of the antibody detection, showing no signal. The right panel depicts the loading control.

Three potential PBMs within XPA-F1 could be identified by in-silico analysis (Figure 4.17). Two putative PBMs are overlapping with XPA’s NLS, and one is located at the ERCC1-binding site. All three show limited consistency with the proposed PBM (2 mismatches) ¹⁴⁷.

In Figure 4.18 a representative PAR overlay of the XPA fragment 2 is shown. The left panel depicts the membrane incubated in polymer. While for H1 a strong signal was observed, protein-bound PAR could neither be detected for BSA nor for XPA-F2. The loading control (right panel) verifies proper loading of proteins.

Figure 4.18: PAR overlay blot with recombinant XPA-F2 fragment. Positive control histone H1, negative control BSA and a fragment of XPA (XPA-F2, aa 60-140) were size separated via SDS-PAGE and subsequently analysed for their non-covalent PAR-binding abilities by far-western blotting. After blotting, the membrane was cut in two identical parts. Membranes were incubated overnight in TBS-T containing 0.2 µM PAR (+PAR) or only in TBS-T (-PAR). Immunostaining of PAR showed no signs of a XPA-F2-PAR interaction. Visualization of His-tag was used as a loading control (Right). One representative blot of two independent experiments is shown.

Figure 4.19: PAR overlay blot with recombinant XPA-F3 fragment. Positive control histone H1, negative control BSA and the minimal DNA-binding site of XPA (XPA-F3, aa 98-211) were size separated via SDS-PAGE and subsequently analysed for their non-covalent PAR-binding abilities by far-western blotting. After blotting, the membrane was cut in two identical parts. Membranes were incubated overnight in TBS-T containing 0.2 µM PAR (+PAR) or only in TBS-T (-PAR). Immunostaining against PAR showed no signs of a XPA-F3-PAR interaction.

Visualization of His-tag was used as a loading control (IB: His-tag) One representative blot of two independent experiments is shown.

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Figure 4.19 depicts a PAR overlay of XPA fragment 3. As for XPA-F2, a strong PAR-binding could be detected for H1, but neither for BSA nor for the MBD of XPA (left panel). The right panel verifies proper protein loading and blotting.

4.3.1.4 Identification and Verification of Novel PBMs in XPA and HERC2

The use of the recombinant XPA fragments revealed the presence of at least one additional PBM in XPA’s N-terminal domain. An in-silico analysis identified two overlapping, putative PBMs, covered by XPA-F1 but not XPA-F2 (Figure 4.16 and Figure 4.17). An in-vitro analysis with a PepSpot membrane, immobilized with these peptides, was conducted to test their ability for interaction with the polymer (Figure 4.20). Both core PBMs showed a strong PAR-binding ability, presumably being responsible for XPA-F1’s PAR-binding. Interestingly, this newly identified PBM overlaps with XPA’s NLS.

In the same approach, putative PAR-binding amino acid sequences within an E3 ubiquitin ligase were tested (Figure 4.20). HERC2 is involved in the ubiquitination and degradation control of XPA and other factors involved in DNA repair and is thus a central regulator of several DNA repair pathways. Two of the three sequences tested here, showed a high degree of consistency with the proposed general PBM, carrying no mismatches, the third contained one mismatch, but is located within a region of several overlapping putative PBMs. The membrane was treated as described above for XPA derived peptides.

All three putative PBMs within HERC2 showed the ability to interact with PAR in a considerable manner.