Cellular characterization of PARP1 variants with altered enzymatic activities

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After genotoxic stress, poly(ADP-ribose) polymerase 1 (PARP1) is activated, leading to poly(ADP-ribosyl)ation (PARylation) of a large variety of target proteins. These targets can be regulated either by covalent modification or non-covalent interactions with poly(ADP-ribose) (PAR). Although the posttranslational modification was extensively studied since its discovery in the 1980s, several aspects remain elusive. Thus, the overall aim of the study was to analyze the relationship between the activity of PARP1, the structure of PAR and the cellular functions regulated by PARylation. Therefore, HeLa PARP1 KO cells were reconstituted with different eGFP-tagged PARP1 variants. This allows the investigation of cellular consequences of different PARP1 variants, without interference of endogenous wild-type PARP1. In the first study (Rank et al., 2016), the suitability of the system for PARylation research was confirmed by using the non-natural PARP1 variants PARP1\L713F and PARP1\E988K. Thereby, it was shown that PARP1\E988K lacks chain elongation activity, rather attaching a single ADP-ribose moiety to target proteins. It was further demonstrated that PARP1\L713F is constitutively active. Both mutants exhibit distinct recruitment kinetics to sites of laser-induced DNA damage and show distinct functional consequences in their cellular patho-physiology. For instance, PARP1\L713F expression was identified to trigger apoptosis, while reconstitution with PARP1\E988K caused a strong DNA damage-induced G2 arrest. After establishing the system as a novel model in PARylation research, two naturally occurring PARP1 variants were analyzed with regards to their potential contribution to human carcinogenesis. It was shown that PARP1 carrying either the single nucleotide polymorphism (SNP) V762A or the inherited mutation F304L, exhibits a reduction in the enzymatic activity. Cells expressing the mutant PARP1 variants further displayed an altered cellular physiology. Taken together, these results strongly support a role of these PARP1 variants in human carcinogenesis. The follow-up study pursues the question if a so called “PAR-code” exists and how chain length and branching frequency of PAR influence cellular functions (Rank et al., manuscript in preparation). To this end, different PARP1 variants producing differently structured PAR were chosen and used for the reconstitution of HeLa PARP1 KO cells. Thereby, the focus was laid on chain length (PARP1\Y986S, short polymer) and the degree of branching (PARP1\Y986H, hyperbranched PAR; PARP1\G972R, hypobranched PAR). Analysis of cellular viability, colony formation capacity and cell cycle status revealed, that especially short and hypobranched PAR is detrimental for the cellular health. Cells expressing the PARP1\G972R variant, exhibit a strongly reduced viability and colony formation capacity as well as a cell cycle arrest in G2 phase. Furthermore, cells expressing PARP1\G972R or PARP1\Y986S displayed higher sensitivities towards genotoxic stress. The recruitment of the different mutants to sites of laser-induced damage was largely impaired for PARP1\G972R and PARP1\Y986S. In addition, PARP1\G972R-reconstituted cells were strongly sensitized towards camptothecin-induced replicative stress, while the effects were less pronounced for PARP1\Y986S. In contrast, PARP1\Y986H showed an almost normal recruitment and response to camptothecin, indicating that the branching frequency is indeed important within the cellular stress response. Furthermore, it was demonstrated that downstream processes like PARP1-dependent transcription and relocalization of XRCC1 rely on a defined PAR structure. Taken together, these results reveal that chain length and branching ratio are essential for the cellular physiology and stress response. Finally, the crosstalk between non-covalent and covalent PARylation was analyzed using p53 as a model substrate (Fischbach et al., 2018). Here, it was shown, that the C-terminal domain (CTD) of p53 is highly important for the PARylation-dependent regulation of p53. It was demonstrated, that binding of p53 to auto-PARylated PARP1 via a highly specific non-covalent interaction renders it a target for covalent PARylation. Importantly, fusing the CTD of p53 to a protein which is normally not PARylated, renders it a target for covalent PARylation as well. It was further shown that PARylation influences the p53-DNA binding properties and has implications on the p53-dependent transcription as well as the interactome. As CTD-like regions are highly enriched in the PARylated proteome, this mechanism might also apply to other PARylation targets. In conclusion the work in this thesis revealed that the here established model, using reconstitution of HeLa PARP1 KO cells with different PARP1 variants, is of great interest for PARylation research in general. Using this model, a significant contribution to the question of the relationship between PARP1, the structure of PAR and the cellular consequences could be provided. It was demonstrated that a tight regulation of PARP1 activity and a defined polymer structure is essential for the cellular physiology and stress response. In addition, crosstalk between covalent and non-covalent PARylation is necessary to achieve a complete regulation and functionality of target proteins.

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ISO 690RANK, Lisa, 2019. Cellular characterization of PARP1 variants with altered enzymatic activities [Dissertation]. Konstanz: University of Konstanz
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@phdthesis{Rank2019Cellu-46686,
  year={2019},
  title={Cellular characterization of PARP1 variants with altered enzymatic activities},
  author={Rank, Lisa},
  address={Konstanz},
  school={Universität Konstanz}
}
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In the first study (Rank et al., 2016), the suitability of the system for PARylation research was confirmed by using the non-natural PARP1 variants PARP1\L713F and PARP1\E988K. Thereby, it was shown that PARP1\E988K lacks chain elongation activity, rather attaching a single ADP-ribose moiety to target proteins. It was further demonstrated that PARP1\L713F is constitutively active. Both mutants exhibit distinct recruitment kinetics to sites of laser-induced DNA damage and show distinct functional consequences in their cellular patho-physiology. For instance, PARP1\L713F expression was identified to trigger apoptosis, while reconstitution with PARP1\E988K caused a strong DNA damage-induced G2 arrest. After establishing the system as a novel model in PARylation research, two naturally occurring PARP1 variants were analyzed with regards to their potential contribution to human carcinogenesis. It was shown that PARP1 carrying either the single nucleotide polymorphism (SNP) V762A or the inherited mutation F304L, exhibits a reduction in the enzymatic activity. Cells expressing the mutant PARP1 variants further displayed an altered cellular physiology. Taken together, these results strongly support a role of these PARP1 variants in human carcinogenesis. The follow-up study pursues the question if a so called “PAR-code” exists and how chain length and branching frequency of PAR influence cellular functions (Rank et al., manuscript in preparation). To this end, different PARP1 variants producing differently structured PAR were chosen and used for the reconstitution of HeLa PARP1 KO cells. Thereby, the focus was laid on chain length (PARP1\Y986S, short polymer) and the degree of branching (PARP1\Y986H, hyperbranched PAR; PARP1\G972R, hypobranched PAR). Analysis of cellular viability, colony formation capacity and cell cycle status revealed, that especially short and hypobranched PAR is detrimental for the cellular health. Cells expressing the PARP1\G972R variant, exhibit a strongly reduced viability and colony formation capacity as well as a cell cycle arrest in G2 phase. Furthermore, cells expressing PARP1\G972R or PARP1\Y986S displayed higher sensitivities towards genotoxic stress. The recruitment of the different mutants to sites of laser-induced damage was largely impaired for PARP1\G972R and PARP1\Y986S. In addition, PARP1\G972R-reconstituted cells were strongly sensitized towards camptothecin-induced replicative stress, while the effects were less pronounced for PARP1\Y986S. In contrast, PARP1\Y986H showed an almost normal recruitment and response to camptothecin, indicating that the branching frequency is indeed important within the cellular stress response. Furthermore, it was demonstrated that downstream processes like PARP1-dependent transcription and relocalization of XRCC1 rely on a defined PAR structure. Taken together, these results reveal that chain length and branching ratio are essential for the cellular physiology and stress response. Finally, the crosstalk between non-covalent and covalent PARylation was analyzed using p53 as a model substrate (Fischbach et al., 2018). Here, it was shown, that the C-terminal domain (CTD) of p53 is highly important for the PARylation-dependent regulation of p53. It was demonstrated, that binding of p53 to auto-PARylated PARP1 via a highly specific non-covalent interaction renders it a target for covalent PARylation. Importantly, fusing the CTD of p53 to a protein which is normally not PARylated, renders it a target for covalent PARylation as well. 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March 8, 2019
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Konstanz, Univ., Diss., 2019
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