Conclusion and Perspectives

During the last 50 years, PARP-1 has been assigned as a caretaker of the genome. This is probably best illustrated by well understood roles in DNA SSBR and DSBR, where PARP-1 serves as a strand break sensor and its PARylation activity facilitates efficient DNA repair. However, PARP-1 has been found to contribute to genomic integrity by several other means as well, such as telomere maintenance, control of cell division, response to replication stress and also NER. In the latter, UV-irradiation has been shown to trigger PARP-1 activity 483,488,559. As mentioned above, DNA strand breaks are strong triggers of PARylation. But in NER, the polymer formation can be observed in the absence of strand breaks and precedes strand incision and potential stimulation by DNA repair intermediates. Still, PARP-1 might directly bind to UV-lesions and upon this binding become activated, as suggested from in-vitro

Figure 5.2: Simplified model of a lesion-specific role of PARP-1 in the response to NER DNA damage. Distinct types of DNA lesions are recognized and removed by the NER pathway. For the repair of these lesions, different sets of DNA repair proteins are requiered for an efficient NER process. PARP-1 facilitates the recognition and repair of UV-light induced DNA lesions, but is dispensable for the removal of bulky BPDE-DNA adducts. Efficiency of NER of different DNA lesions might in turn influence the extent of induced relicative stress in S phase cells.

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experiments ⁵⁵⁹. These experiments demonstrated that the degree of PARP-1 binding and activation is depending on the type of UV-lesion. The more DNA helix distorting 6-4PP served as stronger binding substrates than the moderate kinking CPDs. PARP-1 activation was further enhanced in the presence of DDB2. Stimulation of PARP-1 facilitates subsequent damage recognition and procession by several modes. Accessibility to the DNA lesion site is enhanced by directed chromatin remodelling induced by PARylation of chromatin associated factors, as well as by recruitment of chromatin remodelling proteins such as ALC1 ⁴⁸³. Further, DDB2 protein stability is increased by PARylation, thus preventing its degradation. In turn, this factor further bends the DNA at the lesion site, which as a result increases the recognition and binding of XPC. Herby, the PARP-1-DDB2 axis allows the repair of DNA lesions

Figure 5.3: The role of PARP-1 in response to BPDE-DNA adducts is related to replicative stress, induced by unresolved DNA lesions. A. PARP-1 activity seems to play a minor role in the NER capacity of BPDE-DNA lesions, but still is essential for the efficient cellular response upon BPDE exposure. When unresolved BPDE-adducts are transferred to S phase, relicative stress is induced. PARP-1 binds to and is activated by stalled relication forks. As a result, PARylation slows down replisome progression, thus preventing the accumulation of stalled replication forks. Further, PARP-1 acitvity inhibits untimely resolution of chicken foot structures and facilitates the recruitemnt of MRE11 and induces HR of DSB, resluting from collapsed replication forks. Finally, PARP-1 activity in response to replication stress and DSB formation might influcence energy metabolism by PAR-dependent inhibition of glycolysis. B. As a consequnece, PARylation is an essential factor in the cellular response to bulky DNA lesions, even if not direclty involved in their repair. Deficiency of PARP-1 activty results in enhanced replicative stress, increased numbers of collapsed forks and DSB, and thus higher numbers of accumulated mutations.

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(e.g. CPDs), which were otherwise hardly detected by classic recognition in GG-NER. Downstream of this, PARP-1 interacts with XPA, controlling its functions and thus overall NER, as described within this thesis.

BPDE induces bulky DNA-adducts, but these provoke only moderately DNA distortions ^567,568, which probably provides the cause for their slow repair. Further, the extent of DNA distortion might determine the degree of PARP-1 activation. Since DDB2 does not contribute to the repair of bulky DNA-adducts, thus neither locally kinking the DNA nor interacting with PARP-1, it is questionable if PARP-1 becomes significantly activated through BPDE-DNA lesion. This provides three possible modes of NER response (Figure 5.2). First, DNA lesions which per se strongly distort the DNA (e.g. 6-4PP) are directly recognized by XPC and PARP-1. Second, DNA lesions which only moderately distort the DNA (e.g. CPDs) are recognized and bound by the UV-DDB complex, which enhances the kinking of the DNA. Either by protein-protein interaction with DDB2 or by recognizing the increased helix bending, PARP-1 binds the lesion site. In any case, PARP-1 activation then facilitates downstream NER. In a third situation, weak helix distortion by BPDE-DNA adducts ^567,568, which are further not recognized by the UV-DDB complex, do not trigger PARylation. Thus, readily explaining the insensitivity of NER repair of these lesions to PARP inhibition (4.5.11). Then again, this might implicate a generally poorer NER efficacy, resulting in higher numbers of unresolved DNA lesions transferred to S phase. Here, PARP-1 activity strongly determines the outcome of the resulting replicative stress (Figure 5.3). The role of PARP-1 within replicative stress was recently established. It was shown to bind to and become activated by stalled replication forks ⁴⁰⁶. In turn, PARylation slows down replisomes through the inhibition of DNA polymerases ⁴¹³. This prevents their encounter of lesion sites and thus the accumulation of stalled forks. Additionally, at stalled replication forks PARP-1 stabilizes the ‘chicken foot’ via inhibition of untimely RECQ1-mediated branch migration, thus providing time for DNA lesion removal ^400-402. At the worst, the stalled replication fork collapses, which results in one-ended DNA double-strand breaks. Here, PARP-1 facilitates timely repair by recruitment of MRE11 and stimulation of HR 400,406,407.

This mode of action is neatly pictured in the present work. Unresolved BPDE-lesions induced replicative stress, which was represented by γH2AX signalling in replicating, but not in non-replicating cells. Initial γH2AX levels were comparable between PARP-1 proficient and deficient S phase cells, implying induction of equal amounts of replication stress. Over time (≥ 8 h), differences in damage signalling became more prominent within PARP-1 KO cells, showing strongly increased levels of phosphorylated H2AX. This can be attributed to a divergent outcome from equal amounts of replicative stress. PARP-1 proficient cells readily counteract the replicative stress, prevented strand breakage or efficiently repaired collapsed forks. PARP-1 KO cells however, accumulated more DSB and were less capable to timely repair these by HR. Although the role of PARP-1 in HR is not completely understood, it is believed to facilitate HR even offside of replication stress induced DSBR ^569,570. Further, one-ended DNA double-strand breaks are poor substrates for C-NHEJ. This implicates a further threat for PARP-1 KO cells. As a result, DSBs accumulate and persist over longer periods of time. When eventually being repaired and the cell cycle progressed, increased numbers of mutations accumulated. The accumulation of mutations finally reduces the functionality of the cell. In sum, increased numbers of DSBs and the thereof resulting cell cycle arrest and potential cell death induction, as well as the enhanced genomic instability, manifested in an increased mutation load, can readily explain the strong long-term sensitisation of cells to BDPE treatment upon PARP inhibition. Hence, it would be of interest if cytotoxicity measurements at later time points (> 48 h) would result in increased rates of cell death in PARP-1 deficient cells, or if impaired clonogenic survival is more attributed to a reduction of proliferation.

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The observation of PARylation in response to BPDE exposure led to the initial assumption of an active role of PARP-1 in the NER of BPDE lesions. Since it was found to be dispensable in this, PARylation is not likely to be directly linked to BPDE-DNA lesions per se, but rather to PARP-1 activation at stalled replication forks and resulting DSBs. This assumption is further supported by the time scale of PAR induction. In UV-irradiation experiments PARylation was shown to be induced early on (~5 min) due to direct UV-lesion binding and protein-protein interaction ^483,488. In the present work, significant PAR formation was detected only after 30-60 min. PAR detection in cell cycle synchronized cells could shed further light on the question of the origin of PARP-1 activation. As long as cells do not enter S-phase, BPDE treatment should have limited potential to induce PARylation. Additionally, in-vitro PARylation assays with BPDE treated DNA could provide information on the question on direct activation of PARP-1 by to BPDE-DNA.

In summary, this thesis presents a comprehensive picture of PARP-1 activity in the cellular response to different NER substrates. First, the role of PARP-1 in the NER of UV-photolesions was further resolved. This work provides first functional insights on the consequences of PARP-1 activity, subsequent of initial damage recognition. Hereby, a mutual regulation between PARP-1 and XPA, a factor relevant for both sub-pathways of the NER, was revealed. This offers the possibility of a PARP-dependent fine-tuning of NER in response to UV-irradiation. Moreover, the role of PARP-1 in the cellular response to a bulky DNA-adduct inducing chemical, BPDE, was thoroughly characterized. So far, only little research was performed on NER lesions different from UV-induced DNA damages and the role of PARP-1 herein, and further it was rarely discriminated between different NER lesions types.

Studies dealing with the topic of BPDE and PARP-1 activation often covered only a narrow section of the whole picture 321,326,327. In the present work, a comprehensive model is provided to explain the discrepancy between the observed cellular sensitisation of PARylation deficient cells to BPDE on the one hand and the negligible role of PARP-1 in the NER of BPDE lesions on the other hand. The results of this thesis can likely be used to extent the understanding of other studies with distinct NER triggers, which lack the link between these seemingly opposing observations ⁵⁷¹. This might help to provide a basis to conceive mechanisms underlying several current combinational chemotherapy treatments, including PARP-1 inhibition and induction of NER substrates, such as bulky DNA adduct forming CEES and telozolomide, or DNA intra- and interstrand crosslinking agents as cisplatin, cyclophosphamide and carboplatin ⁵⁷²⁵⁷³.

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Contributions

Collaborations:

[A] D. Gebhard performed the host cell reactivation assay of UV-C irradiated or BPDE-treated reporter plasmids (Figure 4.21B & Figure 4.43)

[B] The PAR size-fractionation depicted in Figure 4.13 was carried out by A. Fischbach.

Master and Bachelor students under the direct supervision of Jan Fischer:

[C] T. Zubel performed the experiments on ROS induction upon BPDE treatment (Figure 4.32), the measurements of PAR formation after exposure to H2O2 and BPDE (Figure 4.35 and Figure 4.36). She contributed to the data on clonogenic survival (Figure 4.38), cytotoxicity (Figure 4.40 & Figure 4.41) and cell cycle analysis upon BPDE treatment (Figure 4.44 & Figure 4.45

& Figure 4.46). Master Thesis, 2015.

[D] K. Jander contributed to the analysis of NAD⁺ levels in response to BPDE exposure (Figure 4.37), the γH2AX detection by western blot (Figure 4.47) and immunofluorescence microscopy (Figure 4.48) and carried out the HPRT forward mutation assay (Figure 4.50). Master Thesis, 2016.

[E] J. Fix performed the site-directed mutagenesis of XPA-PBM_mut1 and XPA-PBM_mu2, and conducted the FRAP experiments (Figure 4.28). She contributed to the analysis of effectiveness of ABT888 treatment (Figure 4.30B), the influence of THF solvent control on cellular health (Figure 4.31A), DNA damage induction by BPDE (Figure 4.33) and the PARP-dependent cellular health analysis using alamarBlue assays (Figure 4.39). Bachelor Thesis, 2014.

Contributions by students of the VTK (2013-2016) as well as assistant researchers (HiWi), both under direct supervision of Jan Fischer, are not listed in detail, but are deeply appreciated (A.

Krüger, W. Naujoks, K. Zwierzynski, I. Trussina, C. Kowarik, P. Palombo, J. Fix, K. Jander).

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Appendix

Table of Figures

Figure 1.1: Reconstructed evolutionary history of the ART superfamily. ... 2 -Figure 1.2: Schematic comparison of the domain architecture of the human PARP (ARTD) family. ... 3 -Figure 1.3: Schematic presentation of the human PARP-1 architecture. ... 4 -Figure 1.4: Model of step-wise assembly of PARP-1 monomer at the sites of DNA single-strand breaks. ... 6 -Figure 1.5: Metabolism of mono- and poly(ADP-ribosyl)ation. ... 7 -Figure 1.6: NAD⁺ metabolism in different cellular compartments. ... 13 -Figure 1.7: Estimated DNA lesions induced by different genotoxic sources and influences of endogenous ...

and exogenous origin. ... 18 -Figure 1.8: Different types of DNA damages are counteracted by distinct DNA repair mechanisms.. ... 20 -Figure 1.9: The nucleotide excision repair pathway. ... 26 -Figure 1.10: Structural model of XPA’s DNA-binding domain. ... 28 -Figure 3.1: Scheme of damage induction. ... 76 -Figure 4.1: Scheme of the XPA fragments spanning regions of interest of XPA. ... 82 -Figure 4.2: Generation of XPA-fragment overexpression constructs. ... 83 -Figure 4.3: Baculovirus titer determination. ... 84 -Figure 4.4: Test expression of His-XPA in High Five cells. ... 84 -Figure 4.5: Test expression of His-XPA-F3 in High Five cells. ... 84 -Figure 4.6: His-XPA purification using HisTrap FF columns in combination with an ÄKTA FPLC. ... 85 -Figure 4.7: His-XPA purification using HiTrap Heparin HP columns in combination with an ÄKTA FPLC. ... 86 -Figure 4.8: XPA-F1 protein purification. ... 86 -Figure 4.9: XPA-F2 protein purification. ... 87 -Figure 4.10: XPA-F3 protein purification. ... 88 -Figure 4.11: Scheme of PARP-1 protein purification. ... 89 -Figure 4.12: Recombinant PARP-1 protein purification and activity test.... 90 -Figure 4.13: Modified silver gel showing in-vitro synthesized PAR. ... 91 -Figure 4.14: XPA binds PAR in a non-covalent manner. ... 92 -Figure 4.15: The XPA-PBM is highly conserved among species. ... 93 -Figure 4.16: PAR overlay blot with recombinant XPA-F1 fragment. ... 94 -Figure 4.17: In-silico identified PBMs in XPA-F1. ... 94 -Figure 4.18: PAR overlay blot with recombinant XPA-F2 fragment. ... 95 -Figure 4.19: PAR overlay blot with recombinant XPA-F3 fragment. ... 95 -Figure 4.20: PAR-overlay assays with custom synthesized peptides were used to test PAR-binding

abilities of putative PBMs. ... 96 -Figure 4.21: PARP activity is essential for efficient repair of UV-C-induced photolesions. ... 97 -Figure 4.22: Electrophoretic mobility shift assay (EMSA) showing XPA binds a biotin-labelled, kinked DNA substrate. ... 97 -Figure 4.23: XPA-PAR interaction inhibits binding of XPA to DNA in-vitro. ... 98 -Figure 4.24: XPA stimulates PARP-1 activity in-vitro. ... 100 -Figure 4.25: PARP inhibition impairs recruitment of XPA-eGFP to sites of laser-induced DNA damage.. .. 101 -Figure 4.26: PARP inhibition delays the recruitment of XPC-eGFP and p53-eGFP to sites of laser-induced DNA damage. ... 103 -Figure 4.27: Sequence of the C-terminal PAR-binding motif of XPA (PBM). ... 105 -Figure 4.28: Fluorescence recovery after photobleaching of wt XPA-eGFP and a PBM-mutated

XPA-eGFP variant. ... 106 -Figure 4.29: Verification of PARP-1 knockout.. ... 106

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Figure 4.30:Stability and temporal effectiveness of the PARP inhibitors ABT888 and PJ34. ... 107 -Figure 4.31:Analysis of the influence of THF on cellular health and the BPDE-induced strand-break

formation. ... 108 -Figure 4.32: ROS formation in HeLa Kyoto cells after BPDE treatment. ... 109 -Figure 4.33: Treatment of HeLa Kyoto cells with BPDE induces bulky DNA adducts. ... 110 -Figure 4.34: PARP inhibition with ABT888 modulates PARP-1 protein levels. ... 111 -Figure 4.35: Time series of PAR formation and degradation after H2O2 damage induction. ... 112 -Figure 4.36: PAR formation in HeLa Kyoto cells after BPDE treatment detected with LC-MS/MS. ... 113 -Figure 4.37: BPDE treatment alters cellular NAD⁺ levels. ... 114 -Figure 4.38:Absence of PARP-1 activity strongly potentiates BPDE’s long-term toxicity. ... 116 -Figure 4.39:Absence of PARP-1 activity protects from BPDE-induced toxicity. ... 118 -Figure 4.40: Representative raw data of annexin V/PI- stained cells obtained by flow cytometry. ... 119 -Figure 4.41: BPDE treatment increases the number of apoptotic and necrotic cells in a

PARylation-independent manner. ... 119 -Figure 4.42: Cellular survival after BPDE treatment is not influenced by nicotinic acid supplementation. 120 -Figure 4.43: PARP activity does not significantly impact on direct BPDE-DNA lesion repair. ... 121 -Figure 4.44 Representative raw data of flow cytometric cell cycle analysis. ... 122 -Figure 4.45: PARP inhibition in the context of BPDE treatment changes the cell cycle phase distribution in HeLa Kyoto cells. ... 123 -Figure 4.46: PARP inhibition potentiates the BPDE-induced cell cycle delay. ... 124 -Figure 4.47 BPDE induces γH2AX signal formation in HeLa Kyoto cells. ... 125 -Figure 4.48: S phase cells immediately responde to BPDE with increased γH2AX signalling. ... 126 -Figure 4.49: PARP-1 deficiency sensitizes cells to BPDE-induced DSB formation. ... 127 -Figure 4.50: PARP inhibition enhances mutagenic potential of BPDE. ... 128 -Figure 5.1: Simplified model of the role of PARP-1 within the NER process. ... 138 -Figure 5.2: Simplified model of a lesion-specific role of PARP-1 in the response to NER DNA damage. ... 145 -Figure 5.3: The role of PARP-1 in response to BPDE-DNA adducts is related to replicative stress,

induced by unresolved DNA lesions. ... 146

-Table of -Tables:

Table 3.1:Thermocycling parameters site-directed mutagenesis. ... 47 -Table 3.2: Thermocycling parameters for XPA-fragment amplification. ... 48 -Table 3.3: Initial (X) and specific (Y) annealing temperatures for each XPA-fragment. ... 49 -Table 3.4: Parameters of HisTrap FF affinity chromatography (His-XPA). ... 54 -Table 3.5:Parameters of HiTrap Heparin HP affinity chromatography (His-XPA). ... 55 -Table 3.6: Parameters of HisTrap FF affinity chromatography (His-XPA-F1). ... 56 -Table 3.7: Parameters of HisTrap FF affinity chromatography (His-XPA-F2). ... 56 -Table 3.8: Parameters of HisTrap FF affinity chromatography (His-XPA-F3). ... 57 -Table 3.9: Preparation of resolving acrylamide gel for SDS-PAGE (8x7 cm²). ... 58 -Table 3.10: Preparation of stacking acrylamide gel for SDS-PAGE (8x1 cm²). ... 59 -Table 3.11: Preparation of in-vitro PARylation reaction mixture. ... 60 -Table 3.12: Composition of modified silver gel. ... 62 -Table 3.13: Pattern used for in-silico PBM search. ... 64 -Table 3.14: Known and putative PBMS of XPA were tested as custom synthesized peptides. ... 64 -Table 3.15: Two putative PBMs of XPA and three of HERC2 were tested with a PepSpot Peptide Array. .... 65 -Table 3.16: Preparation of a 5 % TBE-PAGE for EMSA (8.5cm²). ... 66 -Table 3.17: Source values of MS. ... 69

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Table 3.18: Analyser values for MS. ... 69 -Table 3.19: NAD standard dilution scheme. ... 70 -Table 3.20: Composition of NAD⁺ cycling assay premix. ... 70 -Table 3.21: Click-iT Plus reaction cocktail. ... 74

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Im Dokument The Role of Poly(ADP-Ribosyl)ation in the Molecular and Cellular Response to Nucleotide Excision Repair DNA-Lesions (Seite 158-196)