Generation of Expression Constructs of XPA Fragments

Starting point for the generation of expression constructs was a pSL1180::His-XPA plasmid. Fragments were amplified by PCR and by the use of elongated primers, 5’-end BmtI and 3’-end NotI restriction sites were introduced (Figure 4.2).

PCR products as well as the target vector pVL1392::His were digested (NotI/BmtI) and gel-purified to remove the cut-off products. Subsequently, PCR products were ligated into the pVL1392::His vector. The successful integration and the absence of sequence aberrations was ensured by sequencing.

After baculovirus recombination, Sf9 cells were repeatedly inoculated with the respective virus to amplify the titer. Here, it was taken care not to apply baculovirus with a MOI>1 to avoid accumulation of viruses with mutations in their genome. After 3-4 rounds of amplification, the viral titer was tested by serial dilution of the virus and the infection of a defined number of Sf9 cells.

Virus infected cells, expressing the recombinant protein, were identified by immunofluorescence microscopy (Figure 4.3, representative titer determination of XPA-F3). When obtaining a viral titer in a concentration range of ~1x10⁸ pfu/ml, it was used for overexpression of recombinant proteins.

Amplification of the baculovirus construct carrying the XPA-F4 fragment was not achieved. Expression of the fragment seemed to be cytotoxic.

Figure 4.2: Generation of XPA-fragment overexpression constructs. A. Flowchart showing the process of construct cloning from the PCR of the target region up to the ligation into the baculovirus recombination vector pVL1392. B. Agarose gel visualizing the DNA amplification of the five XPA-fragments.

- 84 - 4.1.2.2 Recombinant Protein Test Expression Test Expression of XPA

Test expressions were performed to optimize the expression conditions of recombinant His-XPA in High Five cells. Cells were inoculated in baculovirus (His-XPA) at MOIs of I and III for up to three days. After the indicted times, cells were harvested, lysed and subject to SDS-PAGE and subsequent immunoblotting (Figure 4.4).

Overexpression of His-XPA could be detected 2 days after infection of cells with the virus and expression levels continued to increase until day

three without showing any signs of degradation. Since a MOI of III induced even higher expression levels than an equal virus-cell ratio, it was decided to perform all following expressions at an MOI of III for 3 days.

Test Expression of XPA-Fragments As described for the overexpression of His-XPA, the optimal expression conditions of the XPA-fragments were identified by test expressions in High Five cells at MOIs of I and III for up to 3 days. As for full-length XPA, all three tested XPA-fragments (F1, F2, F3) showed the highest degree of protein expression at day 3 at a virus MOI of III (Figure 4.5, only XPA-F3 is represented).

Figure 4.3: Baculovirus titer determination. Defined numbers of Sf9 cells were seeded in 96-well plates and inoculated with a baculovirus of unknown titer. After three days, cells were fixed and an immunofluorescence analysis of recombinant protein expressing cells was conducted with target protein specific antibodies. Depicted is a representative image of the forth amplification of XPA-F3 baculovirus at a dilution of 1:10. Scale bar represents 50 µm.

Figure 4.4: Test expression of His-XPA in High Five cells. At an MOI of I or III cells were inoculated for up to three days in baculovirus. Cells were lysed and His-XPA expression was detected by immunoblotting.

Figure 4.5: Test expression of His-XPA-F3 in High Five cells. At an MOI of I or III cells were inoculated for up to three days in baculovirus. Cells were lysed and XPA-F3 expression was detected by immunoblotting.

- 85 - 4.1.2.3 Purification of Recombinant Proteins Purification of Full-Length His-XPA

For the purification of His-XPA, an ÄKTA FPLC based two-step purification process was established.

First, it was taken advantage of the poly(His)-tag of XPA using IMAC with HisTrap FF columns. XPA was further purified using HiTrap Heparin HP columns in a second affinity chromatography, in which XPA’s DNA-binding ability was exploited.

His-XPA expressing High Five cells were lysed and genomic DNA was fragmented by sonification.

Cell extracts were precleared by ultracentrifugation and subsequent filtration, diluted in purification buffer containing 5 mM imidazole, and loaded on a HisTrap FF column (Figure 4.6A).

Despite a significant portion of insoluble recombinant XPA (Figure 4.6B, cell debris pellet), sufficient amounts of protein could be found in the cell extract, which very efficiently bound to the column material. Subsequently, the column was rinsed with elution buffer with increasing concentrations of imidazole. XPA was eluted from the column at 200 mM imidazole. The majority of the protein could be found in the fractions A7-12 (Figure 4.6B).

The XPA containing fractions were pooled, diluted in XPA purification buffer C containing 40 mM NaCl, and loaded via ÄKTA FPLC on HiTrap Heparin HP columns (Figure 4.7A). Again, His-XPA was nearly completely trapped on the purification material and no recombinant protein could be observed in the flow through. The column was rinsed with elution buffer containing increasing concentrations of NaCl. XPA eluted at 400 mM NaCl and the majority of the protein could be found in fractions D4-E6 (Figure 4.7B). Purity of the purified protein varied in the different fractions between 50-95 % and were separately processed in the following.

Figure 4.6: His-XPA purification using HisTrap FF columns in combination with an ÄKTA FPLC. After clarification of the crude cell extract, the lysate was subject to a first IMAC purification step with HisTrap FF columns. A.

Chromatogram of the IMAC purification. His-XPA eluted from the column at 200 mM imidazole. B. Coomassie-stained SDS-PAGE (4-20 %) of different preparation steps and elution fractions. Majority of His-XPA (42 kDa) could be found in fractions A7 to A12.

- 86 - Purification of His-XPA Fragments

Three fragments of XPA were purified in the course of this thesis: the N-terminal fragment XPA-F1, the central minimal DNA-binding domain containing XPA-F3 and XPA-F2, covering a region overlapping the C-terminus of F1 and the N-terminus of F3. His-tagged fragments were overexpressed in High Five cells for three days (MOI III), cells were pelleted and flash frozen for storage. Cell pellets Figure 4.7: His-XPA purification using HiTrap Heparin HP columns in combination with an ÄKTA FPLC. After a first HisTrap FF IMAC purification, pooled elution fractions containing His-XPA were subject to a second purification step with heparin sepharose. A. Chromatogram of the affinity purification. His-XPA eluted from the column at 400 mM NaCl. B. Coomassie-stained SDS-PAGE (4-20 %) of input, flow through and elution fractions.

Majority of His-XPA (42 kDa) could be found in fractions D4 to E6.

Figure 4.8: XPA-F1 protein purification. Fragment 1 was purified in two subsequent steps, IMAC and SEC. A.

Chromatogram of IMAC with HisTrap FF (Ni sepharose) columns. Crude cell lysate was cleared and subject to a FPLC-based purification. B. Coomassie-stained SDS-PAGE (4-20 %) of A. Majority of XPA-F1 could be found in elution fractions C11-D10 (17.5 kDa). C. Size exclusion chromatography was used to further purify XPA-F1. The arrow marks the run volume at which F1 eluted from the column. D. Coomassie-stained SDS-PAGE (4-20 %) of C. F1 eluted with a high degree of purity form the SEC.

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were lysed, the DNA was sheared by sonification and the cell extract was cleared by ultracentrifugation and subsequent filtration. The filtrate was diluted in XPA purification buffer (5 mM imidazole) and purified via ÄKTA FPLC.

XPA-F1 Purification:

Due to the N-terminal hexahistidine tag, XPA-F1 was first purified by affinity chromatography with Ni sepharose. The cleared cell extract was loaded via an ÄKTA FPLC on HisTrap FF columns and unspecific, weakly bound proteins were removed by washing with elution buffer containing low concentrations of imidazole. Elution of XPA-F1 was achieved by rinsing the column with 500 mM imidazole. The majority of the recombinant protein could be found in fractions C11-D10 (Figure 4.8A and B). Recombinant protein-containing elution fractions were pooled, concentrated, and further purified by size exclusion gel chromatography (SEC). During SEC, proteins are separated according to their molecular mass, large proteins eluting earlier than smaller ones. Identification of the elution peak in SEC chromatogram was difficult (Figure 4.8C, see arrow), but the target protein containing fractions

Figure 4.9: XPA-F2 protein purification. Fragment 2 was purified in two subsequent steps, IMAC and SEC. A.

Chromatogram of IMAC with HisTrap FF (Ni sepharose) columns. Crude cell lysate was cleared and subject to a first FPLC-based purification. B. Coomassie-stained SDS-PAGE (4-20 %) of A. Majority of XPA-F2 could be found in elution fractions B8 and B7 (18.5 kDa). C. Size exclusion chromatography was used for further purification.

The arrow marks the run volume at which F2 eluted from the column. D. Coomassie-stained SDS-PAGE (4-20 %) of C. F2 eluted with a high degree of purity from the SEC column.

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could be determined by Coomassie-stained SDS-PAGE and immunoblotting (Figure 4.8D and Figure 4.16 right panel). While the actual molecular mass of the fragment was calculated to be 12.5 kDa, the apparent molecular weight was 17.5 kDa. Purity of the XPA-F1 containing fractions (E6-F7) extended from 30-95 %. Elution fractions were further processed separately according to their degree of purity.

XPA-F2 Purification:

XPA-F2 was purified as described for F1, with modifications (Figure 4.9). Cell extracts were cleared by centrifugation and filtration, and were loaded via an ÄKTA FPLC on HisTrap FF columns. The recombinant protein bound with a high efficiency to the column, while the majority of the protein impurities could be found in the flow through. Washing the column with 50 mM imidazole already eluted a relevant portion of the XPA fragment, but the majority was only eluted when flushing the column with 350 mM imidazole (Figure 4.9A). The eluates were subject to SDS-PAGE and subsequent Coomassie staining, identifying B8 and B7 as the fractions containing the majority of XPA-F2 (Figure 4.9B). All XPA-F2 containing fractions were pooled, concentrated and further purified by size exclusion chromatography. The fractions containing the recombinant protein (Figure 4.9C, arrow) were identified by SDS-PAGE, Coomassie staining and immunoblotting (Figure 4.9D and Figure 4.18).

Majority of XPA-F2 could be found in the fractions 2H5-2H9 with a purity ~85 %, which were pooled and concentrated. The actual molecular mass of XPA-F2 was identified as 18.5 kDa, while the apparent molecular mass was calculated to be 11.5 kDa.

XPA-F3 Purification:

The cleared cell lysate from XPA-F3 expressing High Five cells was loaded on a HisTrap FF column via an ÄKTA FPLC. The recombinant protein bound efficiently to the chromatography media, while

Figure 4.10: XPA-F3 protein purification. Fragment 3 purification was performed by affinity chromatography with HisTrap FF (Ni sepharose) columns. A. Chromatogram of IMAC with HisTrap FF columns. Crude cell lysate was cleared and subject to FPLC-based purification. B. Coomassie-stained SDS-PAGE (4-20 %) of A. Majority of XPA-F3 can be found in elution fractions B5-C4 (16 kDa).

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most of the protein impurities could be found in the flow through (Figure 4.10A). A three step imidazole gradient was applied for further purification. First, a flat gradient up to 25 mM imidazole was run to wash out unspecifically bound protein. Second, a steeper gradient up to 250 mM imidazole was run.

Finally, a third gradient up to 500 mM imidazole was applied. XPA-F3 eluted efficiently at about 100-150 mM imidazole. Fractions were subject to SDS-PAGE, Coomassie staining and immunoblotting (Figure 4.10B and Figure 4.19). After this step, a high degree of purity (^~90 %) of the recombinant protein was reached and no further purification was required. The actual molecular mass of XPA-F3 was identified as 17 kDa, while the apparent molecular mass was calculated to be 16 kDa.

4.1.3 PARP-1

4.1.3.1 PARP-1 Overexpression

Expression of recombinant PARP-1 was performed in Sf9 insect cells as described previously ¹⁵². Sf9 cells were inoculated in baculovirus at a MOI of I for 3 days. After that time, recombinant protein expression maximized but minimal protein degradation was maintained (data not shown). Cells were harvested, pelleted and flash frozen in liquid nitrogen for storage.

4.1.3.2 PARP-1 Purification

Purification of PARP-1 was performed by gravity flow chromatography. After cell lysis, debris was removed by ultracentrifugation. Treatment with protamine sulphate precipitated cellular DNA, while two subsequent salting-out steps with ammonium sulphate separated recombinant PARP-1 from proteins with distinct solubility. The purification process was followed by SDS-PAGE and subsequent Coomassie staining or immunoblotting to monitor the amount of PARP-1 in the single fractions (Figure 4.12).

Next, gel filtration was used for desalting and rebuffering of the PARP-1 containing solution. Here, most of the recombinant protein eluted early on, with the bulk of PARP-1 being present in the first elution fraction (Figure 4.12A). PARP-1 containing fractions (E1-E3) were pooled and subject to an affinity purification step using dsDNA cellulose (Figure 4.12B). Although some protein loss could be observed in the flow through, a considerable amount of PARP-1 bound to the purification material. While unspecifically bound proteins were removed, the PARP-dsDNA cellulose interaction was not affected by the following washing steps.

PARP-1 was eluted in three subsequent elution steps with a good degree of purity, which increased with each elution fraction (E2<E3<E3). Each eluate was separately dialyzed against storage buffer. Protein concentrations were determined (300-400 ng/ml) and enzyme activity was tested in an in-vitro PARylation assay (Figure 4.12C). Reactions were carried out with 5 nM PARP-1 for 60 s at 30°C. PAR was slot-blotted on a nylon membrane and immunochemical detection was performed to compare the levels of formed polymer of the elution fractions J1E2-J1E4 with a previously purified PARP-1 batch (R5). At least two fractions indicated for a higher PARylation activity (J1E2 and J1E4). Fraction J1E4,

Figure 4.11: Scheme of PARP-1 protein purification. PARP-1 was purified in three steps: protein precipitation, gel filtration and an affinity purification.

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showing the highest degree of purity and satisfying activity, was used for all biochemical analysis performed in this thesis, while the other fractions were used for in-vitro PAR synthesis.

Figure 4.12: Recombinant PARP-1 protein purification and activity test. A. After protein precipitation, the next step for PARP-1 purification was gel filtration with self-prepared sephadex G-100 sf columns. A Coomassie-stained SDS-PAGE (left) and the corresponding immunoblot for PARP-1 (right) are shown. The crude cell lysate, resuspended cell debris pellets, the clarified cell lysate and the eluates were size separated. PARP-1 (113 kDa) could be found in eluate 1-4. B. IMAC was performed with the pooled fractions for further purification. PARP-1 bound efficiently to dsDNA cellulose and could be eluted in the fractions E2-E4 with a high degree of purity. C.

Eluted PARP-1 was dialysed and tested for their PARylation capacity in an in-vitro PARylation assay. The reaction mixture was subject to a slot-blot and PAR was immunochemically detected with the PAR specific antibody 10H.

Comparison with a previously purified PARP-1 batch showed that each of the eluted PARP-1 protein fractions was active and formed polymer to a satisfying degree.

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4.2 In-Vitro PAR Synthesis

For many biochemical assays performed in this thesis purified PAR, size fractionated as well as unfractionated, was required. PAR synthesis was performed in an in-vitro PARylation assay.

Therefore, recombinant PARP-1 protein was mixed with high concentrations of NAD⁺ and an activating duplex oligonucleotide. By varying the amount and proportional distribution of added histones (H1 &

H2A) or by altering the reaction time, the complexity and length of the formed polymer could be influenced as required. Quantity and purity of synthesized PAR was controlled by UV absorbance (A258 and A280, respectively) and the use of the molar absorption coefficient of mono(ADP-ribose) of 13,500 M^-1 cm^-1. Since the quantity was defined by the absorption coefficient of mono(ADP-ribose), concentrations given in this thesis for unfractionated polymer always refer to ADP-ribose moieties. Concentrations for size fractionated PAR on the other hand, were recalculated per PAR chain (average size of the fraction). Besides controlling the quality of the PAR synthesis by analysis of the UV-absorption, each PAR batch was subject to a modified silver gel. The polymer was size separated on a 20 % TBE-PAGE and subsequently visualized by silver staining (Figure 4.13).

Previous studies suggested a relevance of the quality

of PAR, in respect of length and branching, for its role in orchestrating cellular mechanisms ^152,153. While some proteins seem to interact with the polymer independent of its chain length, others, like XPA, showed a high degree of specificity, strongly preferring longer PAR chains for binding. Anion exchange chromatography via HPLC with a multi-step gradient up to 1 M NaCl was used to size separate PAR chains, only varying by a few ADP-ribose units (Figure 4.13). With the size fractionated PAR, more specific material was at hand to perform precise assays and reactions.

4.3 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

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

Im Dokument The Role of Poly(ADP-Ribosyl)ation in the Molecular and Cellular Response to Nucleotide Excision Repair DNA-Lesions (Seite 96-0)