The Role of Poly(ADP-Ribosyl)ation in the Molecular and Cellular Response to Nucleotide Excision Repair DNA-Lesions
Dissertation
zur Erlangung des akademischen Grades des
Doktors der Naturwissenschaften (Dr. rer. nat.)
an der Universität Konstanz
Mathematisch-Naturwissenschaftliche Sektion Fachbereich Biologie
vorgelegt von
Jan Fischer
Tag der mündlichen Prüfung: 29. Juni 2016
Referenten: Prof. Dr. Alexander Bürkle (Gutachter und Prüfer) Prof. Dr. Tanja Schwerdtle (Gutachter und Prüfer) Prof. Dr. Christof Hauck (Prüfer)
Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-0-348334
II
III
cellular and molecular response to BPDE-DNA lesions. Manuscript in preparation.
JM Fischer, O Popp, D Gebhard, S Veith, A Fischbach, S Beneke, A Leidensdorfer, J Bergemann, M Scheffner, E Ferrando-May, A Mangerich, A Bürkle (2014): Poly(ADP-ribose)-mediated interplay of XPA and PARP1 leads to reciprocal regulation of protein function. The FEBS journal 281, 3625-3641, doi:10.1111/febs.12885.
Other Publications:
L Rank, S Veith, E Gwosch, J Demgenski, M Ganz, M Jongmans, C Vogel, A Fischbach, S Bürger, A Stier, JM Fischer, C Renner, M Schmalz, S Beneke, M Groettrup, R Kuiper, A Bürkle, E Ferrando- May, A Mangerich: Structure-function relationships of natural and artificial PARP1 variants in reconstituted HeLa PARP1 knock-out cells. Submitted in Nucleic Acids Research.
AJ Renz, HM Gunter, JM Fischer, H Qiu, A Meyer, S Kuraku (2011): Ancestral and derived attributes of the dlx gene repertoire, cluster structure and expression patterns in an African cichlid fish. Evodevo 2, 1, doi:10.1186/2041-9139-2-1.
Presentations at Scientific Conferences:
Poster Presentation:
10th Quinquennial Conference on Responses to DNA Damage: From Molecule to Disease. – Egmond aan Zee, Netherlands 2016
Konstanz Symposium Chemical Biology. – Konstanz, Germany 2015
3. Tagung der Experimentellen und Klinischen Pharmakologen und Toxikologen in Baden- Württemberg. – Tübingen, Germany 2015
SFB969 Review. – Konstanz, Germany 2015 KoRS-CB Retreat. – Gültstein, Germany 2015
Two days of Proteostasis in Konstanz. – Konstanz, Germany 2014 KoRS-CB Retreat. – Bad Herrenalb, Germany 2014
German-French DNA Repair Meeting on Epigenetics and Genome Integrity. – Strasbourg- Illkirch, France 2013
KoRS-CB Retreat. – Gültstein, Germany 2013
Two days of Proteostasis in Konstanz. – Konstanz, Germany 2012 6th PARP Regio Meeting 2012. – Aachen, Germany 2012
Eleventh Symposium on Neurobiology and Neuroendocrinology of Aging. – Bregenz, Austria 2012
IV
13th Biennial Conference of the DGDR. – Mainz, Germany 2014 SFB969 Retreat. – Bräunlingen, Germany 2013
KoRS-CB Student Seminar. – Konstanz, Germany 2013
Fellowships, Stipends and Awards:
2012-2016: Fellow of the Konstanz Research School Chemical Biology (KoRS-CB).
2012-2015: Member of the Sonderforschungsbereich 969 (SFB969).
2014: Recipient of a Travel Scholarship (‘13th Biennial Conference of the DGDR’).
2009-2010: Recipient of a Fulbright Scholarship.
Attended Courses of the Graduate School Chemical Biology (KoRS-CB):
Gene Expression and Protein Purification Strategies.
Biomedicine. Flow Cytometry and Fluorescence Activated Cell Sorting.
Determination of Macromolecular Structures.
Proteomics.
Electron Microscopy.
The Power of Cutting Edge Mouse Genetics.
Matlab.
Patents in Real Life.
Additional Courses Attended at the University of Konstanz:
European Business Competence (EBCL-B).
‘Laboratory Animal Sciences’ after guidelines of FELASA-Cat. B.
Introduction into Safety Difficulties of Genetic Engineering.
(Approved training according to the German § 15 GenTSV) Photoshop Workshop.
V
Zusammenfassung
Poly(ADP-ribosyl)ierung (PARylation) ist eine komplexe und reversible posttranslationale Modifikation. Diese Reaktion wird katalysiert von der Enzymklasse der Poly(ADP- ribosyl)polymerasen (PARPs) und nimmt auf diese Weise Einfluss auf eine Reihe anderer Proteine, reguliert und koordiniert sowohl ihre Funktionen als auch ihre räumliche Verteilung innerhalb der Zelle.
Hierdurch spielt PARP-1 eine zentrale Rolle in einer Vielzahl von zellulären Vorgängen, wie zum Beispiel der DNA Reparatur, der strukturellen Organisation des Chromatins, der Transkription von Genen und der Kontrolle des Zelltodes. Die zugrunde liegenden Mechanismen von PARP-1 in der Reparatur von DNA Einzel- sowie Doppelstrangbrüchen (SSB und DSB) wurden umfassend untersucht und können zu weiten Teilen nachvollzogen werden. Im Gegensatz dazu ist die Rolle von PARP-1 in der Nukleotidexzisionsreparatur (NER) weit weniger gut verstanden. Der NER ist ein DNA- Reparaturweg, hauptverantwortlich für die Beseitigung einer ganzen Reihe unterschiedlicher DNA Läsionen. Diese Läsionen haben gemein, dass sie die native Konformation der DNA stören, sie biegen oder die lokale Basenpaarung destabilisieren. Verursacht werden solche Konformationsveränderungen unter anderem durch UV-Photoprodukte oder aber durch Chemikalien-induzierte große DNA-Addukte.
Das Protein XPA übernimmt innerhalb des NERs eine zentrale und koordinierende Funktion und ist involviert im Aufbau des Prä-Inzisionskomplexes, der Verifizierung des DNA Schadens und der korrekten Platzierung der für die erfolgreiche Entfernung des Schadens nötigen Endonukleasen.
Obwohl XPA eines der ersten NER Proteine war, für welches eine nicht-kovalente Bindung von PAR nachgewiesen werden konnte, ist diese Interaktion, sowie die daraus resultierenden funktionalen Konsequenzen kaum untersucht. Im Zuge dieser Thesis konnte ein förderlicher Einfluss der PARP-1 Aktivität auf die NER-Reparaturkapazität von UV-Photoprodukten verifiziert, sowie eine wechselseitige Regulation mit XPA aufgedeckt werden. Das bereits bekannte PAR-Bindemotiv (PBM) von XPA wurde im Detail charakterisiert und die für die PAR-Bindung essentiellen Aminosäuren identifiziert. Weiterführend konnte ein bisher unbekanntes PBM im N-terminalen Abschnitt von XPA lokalisiert werden, welches mit anderen bedeutenden XPA-Domänen überlappt. Mittels biochemischer und zellbiologischer Versuche wurden die funktionalen Konsequenzen der XPA-PAR Interaktion weiter analysiert. Hierbei konnte eine reziproke Regulation zwischen PARP-1 und XPA aufgedeckt werden. Die Bindung von XPA an PAR beeinträchtigte XPAs DNA-Bindefähigkeit und zwar in Abhängigkeit von der PAR-Kettenlänge. Zudem konnte gezeigt werden, dass eine effiziente Rekrutierung von XPA, sowie anderen reparaturanverwandten Proteinen, zu laserinduzierten DNA Läsionen eine aktive PARylierung voraussetzt. Auf der andern Seite konnte mittels in-vitro PARylierungs Assays gezeigt werden, dass XPA die Aktivität von PARP-1 sogar in der Abwesenheit von DNA stimulieren kann, vermutlich durch eine direkte Protein-Protein Interaktion. Schließlich wurden erste Experimente durchgeführt zur Generierung und funktionalen Charakterisierung einer PAR-bindedefizienten XPA Mutante.
Zusammenfassend konnte in dieser Arbeit gezeigt werden, dass XPA und PARP-1 in der Lage sind sich in ihren Funktionen wechselseitig zu regulieren. Diese wechselseitige Interaktion trägt vermutlich zu einer Feinabstimmung bei, die zur optimalen Erkennung, Verifizierung und Reparatur von UV- induzierten Photoläsionen benötigt wird.
Auf der anderen Seite weisen die Untersuchungen mit einem weiterem NER Substrat, Benzo[a]pyren- 7,8-dihydroxy-9,10-epoxid (BPDE), auf eine hiervon unterschiedliche Rolle von PARP-1 in der DNA Schadensantwort hin. Mittels ‚Isotope-Dilution‘ Massenspektroskopie, sowie enzymatischer ‚NAD+ cycling‘ Assays konnte eine zeit- und BPDE dosisabhängige PARP-1 Stimulation detektiert werden.
Diese scheint jedoch nicht im direkten Zusammenhang mit der Entfernung der DNA Addukte zu stehen.
VI
jedoch auf eine funktionale Relevanz der PARP-1 Aktivität in der zellulären Antwort auf BPDE- induzierten genotoxischen Stress hin. Obgleich die zytotoxische Wirkung von BPDE in den ersten 48 Stunden nach Behandlung nicht durch die Aktivität von PARP-1 beeinflusst wurde, konnte auf längere Sicht eine deutliche Sensibilisierung in PARylation-defizienten Zellen festgestellt werden. Die Behandlung mit BPDE verursachte einen markanten S und G2 Phasenarrest, welcher durch die Inhibition von PARP-1 weiter verstärkt wurde. Dies deutet darauf hin, dass nicht-reparierte BPDE- DNA Addukte zu replikativem Stress in S Phase Zellen führen. Unterstützend hierzu konnte zeitnah nach der BPDE Behandlung ein deutliches DNA Schadenssignal (γH2AX) in S Phase aber nicht in nicht-replizierenden Zellen, detektiert werden. Die Folgen des replikativen Stresses sind hierbei abhängig von der PARP-1 Aktivität. In PARP-1-defizienten Zellen führte die BPDE Behandlung zu einer deutlich verstärkten Bildung und Persistenz von DNA DSB. Im Einklang damit konnte mittels eines ‚HPRT forward-mutation‘ Assays nachgewiesen werden, dass die Inhibierung von PARP-1 das mutagene Potential von BPDE verstärkt und somit eine Anreicherung von Mutationen im Genom bewirkt. Die hierdurch beeinträchtigte zelluläre Funktionalität kann als Konsequenz die Langzeitsensibilisierung von PARylation-defizienten Zellen auf BPDE erklären.
Zusammenfassend beschreibt die vorliegende Studie eine signifikante Rolle von PARP-1 in der Schadensantwort auf BPDE-induzierten genotoxischen Stress. Der Mechanismus, der diesem Einfluss zugrunde liegt ist jedoch ein anderer als beschrieben für die Reaktion auf UV-Photolesionen. In die Reparatur von UV-Schäden ist PARP-1 direkt involviert und fördert, unter anderem durch die wechselseitige Regulation mit dem zentralen NER-Koordinator XPA, die Prozessierung und Entfernung der Läsionen aus dem Genom. Auf der anderen Seite scheint die Aktivität von PARP-1 für die Reparatur von BPDE-DNA Addukten entbehrlich zu sein. Abseits einer direkten Rolle in der Reparatur dieser Läsionen, erfüllt PARP-1 einen essentiellen und fördernden Einfluss auf die DNA- Schadensantwort. PARP-1 limitiert die Auswirkungen des BPDE-induzierten replikativen Stresses und der daraus resultierenden DNA Strangbrüche sowie der genomischen Instabilität. All dies deutet auf eine läsionsspezifische Rolle von PARP-1 in der Reparatur von NER zughörigen DNA Schäden hin.
VII
Abstract
Poly(ADP-ribosyl)ation (PARylation) is a complex and reversible posttranslational modification catalyzed by poly(ADP-ribose)polymerases (PARPs), which thereby orchestrate other proteins’
functions and localizations. PARP-1 plays significant roles in many different cellular aspects, such as DNA repair, chromatin remodeling, gene transcription and regulation of cell death. While a role of PARP-1 in DNA single-strand break repair (SSBR) and double-strand break repair (DSBR) is firmly established, its role in nucleotide excision repair (NER) is less well understood. NER is a versatile DNA repair pathway involved in the removal of a multitude of different helix distorting DNA lesions, such as UV-light-induced photolesions or bulky DNA adducts. Herein, XPA can be considered as central coordinator of the NER machinery, involved in the assembly of the preincision complex, the verification of DNA lesions and correct placement of the endonucleases essential for successful damage removal.
Although XPA being one of the first NER factors described to interact with PAR, this interaction and its functional consequences are poorly understood. In the course of this thesis, the beneficial impact of PARP-1 activity on the NER capacity of UV-photoproducts was verified and its mutual regulation with XPA was described in detail. The previously identified PAR-binding-motif (PBM) of XPA was further characterized and amino acids essential for the non-covalent interaction with PAR were identified.
Additionally, a second, so far undescribed PBM could be located within the N-terminal part of XPA, overlapping with several important domains. The functional consequences of the XPA-PAR interaction were addressed in biochemical and cellbiological approaches. Interestingly, a reciprocal regulation between PARP-1 and XPA could be identified. XPA-PAR-binding interfered with its DNA-binding abilities in a PAR chain length-dependent manner. Efficient recruitment of XPA, as well as other repair- related proteins, to sites of laser-induced DNA damage was shown to be dependent on active PARP-1 and were delayed upon PARP inhibition. On the other hand, in-vitro PARylation assays suggested a direct protein-protein interaction between these two proteins, leading to a stimulation of PARP-1 activity even in the absence of damaged DNA. Finally, first steps were undertaken to generate, and functionally characterize a PAR-binding deficient XPA mutant.
In conclusion, these results revealed that XPA and PARP-1 are able to regulate each other in a reciprocal and PAR-dependent manner. It can be assumed that the mutual control is part of a fine-tuning mechanism mediated by PARP-1 to efficiently recognize, verify and process UV-induced photolesions.
On the other hand, when proceeding studies with another NER substrate, benzo[a]pyrene-7,8- dihydroxy-9,10-epoxide (BPDE), a different role of PARP-1 in the damage response could be revealed.
PAR formation after BPDE exposure could be proven to occur in a time- and dose-dependent manner by using isotope dilution mass spectrometry and an enzymatic NAD+ cycling assay. This observation seems not to be directly related to the removal of bulky DNA adducts. Here, the analysis of the NER capacity in the absence of PARP activity did not implicate a significant role of PARP-1 in the repair of BPDE-DNA adducts. On the other hand, cellular studies such as analysis of cell proliferation, cell cycle, and clonogenic survival demonstrated a functional relevance of PARP activity in the BPDE-induced genotoxic stress response. While the cytotoxic impact of BPDE during the first 48 hours after treatment was not affected when PARylation activity was impaired, PARP inhibition strongly sensitized cells on longer terms. BPDE itself caused a marked S and G2 phase arrest, which was further potentiated in the absence of active PARP-1 protein. This points towards unresolved BPDE-DNA lesions, triggering replicative stress during S-phase. In line with this, DNA damage signaling (γH2AX) could be observed early on after BPDE treatment in replicating cells but not in non-S phase cells. The outcome of this replicative stress was strongly dependent on the cells’ proficiency for PARylation. In PARP-1 deficient cells, BPDE exposure resulted in enhanced formation and persistence of DNA DSBs. In line with this,
VIII deficiency.
In sum, in the course of this study a role of PARylation in BPDE-induced genotoxic stress was established. This role was shown to be distinct from the function of PARP-1 established in response to UV-photolesions. In this, PARP-1 positively affects NER capacity and is directly involved in the repair of UV-photoproducts, including by mutual regulation with the NER coordinator XPA. On the other hand, PARP-1 seems to be dispensable for the NER-mediated removal of bulky DNA-adducts induced by BPDE. But even without direct linkage to lesion repair, PARP-1 still has a beneficial effect on the DNA damage response. Here, PARP-1 seems to reduce the impact of induced replicative stress, resulting DNA strand breaks and genomic instability. All this points towards a lesion-specific role of PARylation in the repair of DNA damage related to NER.
IX
Table of Content
1 Introduction ... - 1 -
1.1 The ADP-Ribosylation System ... - 1 -
1.1.1 The ART-Superfamily ... - 1 -
1.2 The PARP Superfamily ... - 3 -
1.2.1 Classification and Organisation of PARPs ... - 3 -
1.2.1.1 Evolutionary Clustering ... - 3 -
1.2.1.2 The PARP Family ... - 4 -
1.2.1.3 Structure of PARP-1 ... - 4 -
1.2.1.4 MARylation and PARylation Activities of PARP Family Members... - 5 -
1.2.2 PAR Metabolism ... - 5 -
1.2.2.1 PAR Anabolism ... - 5 -
1.2.2.2 PAR Catabolism ... - 9 -
1.2.3 Non-Covalent PAR-Binding Modules ... - 10 -
1.2.4 Biological Functions of PARP-1 ... - 11 -
1.2.5 Therapeutic Approaches and the Concept of Synthetic Lethality ... - 16 -
1.3 DNA Damage and DNA Damage Response ... - 17 -
1.3.1 Mechanisms Resulting in Genomic Instability ... - 17 -
1.3.1.1 Benzo[a]pyrene and its Reactive Epoxide Metabolite ... - 19 -
1.3.2 Mechanisms of Maintaining Genomic Integrity ... - 19 -
1.3.3 Direct Damage Removal ... - 20 -
1.3.4 DNA Mismatch Repair ... - 21 -
1.3.5 Base Excision Repair ... - 21 -
1.3.6 Single-Strand Break Repair ... - 22 -
1.3.7 DNA Replication Stress Response ... - 22 -
1.3.8 Double-Strand Break Repair ... - 23 -
1.3.9 Nucleotide Excision Repair ... - 25 -
1.3.10 Xeroderma Pigmentosum, Complementation Group A ... - 27 -
1.3.11 NER Associated Disorders ... - 29 -
2 Motivation ... - 30 -
3 Material and Methods... - 31 -
3.1 Material... - 31 -
3.1.1 Cell Lines ... - 31 -
3.1.2 Cell Culture Media ... - 31 -
3.1.3 Enzymes ... - 31 -
X
3.1.4 Antibodies ... - 32 -
3.1.5 Chemicals and Reagents ... - 32 -
3.1.6 Material and Kits ... - 34 -
3.1.7 Plastics ... - 35 -
3.1.8 Glassware ... - 35 -
3.1.9 Buffers and Solutions ... - 35 -
3.1.10 Laboratory Equipment ... - 40 -
3.1.11 Plasmids ... - 41 -
3.1.12 Oligonucleotides ... - 41 -
3.1.13 Software ... - 43 -
3.2 Methods ... - 44 -
3.2.1 Cell Culture ... - 44 -
3.2.1.1 General Aspects of Cell Culture ... - 44 -
3.2.1.2 Freezing of Cells ... - 44 -
3.2.1.3 Thawing of Cells ... - 44 -
3.2.1.4 Insect Cell Culture ... - 44 -
3.2.1.5 Mammalian Cell Culture ... - 45 -
3.2.2 Molecular Cloning ... - 45 -
3.2.2.1 General Aspects ... - 45 -
3.2.2.2 Preparing Chemically Competent DH5α Cells, of LB Agar and Agar Plates ... - 45 -
3.2.2.3 Polymerase Chain Reaction (PCR) ... - 45 -
3.2.2.4 Transformation of Chemically Competent DH5α ... - 45 -
3.2.2.5 Plasmid DNA Purification ... - 46 -
3.2.2.6 Analytical an Preparative Plasmid DNA Digestion ... - 46 -
3.2.2.7 Agarose Gel Electrophoresis... - 46 -
3.2.2.8 DNA Gel Extraction ... - 46 -
3.2.2.9 Insert-Vector DNA Ligation ... - 47 -
3.2.2.10 Site-Directed Mutagenesis (SDM) ... - 47 -
3.2.2.11 Cryoconservation of DH5α Cells ... - 47 -
3.2.2.12 PAR-Binding Motif Alignment ... - 48 -
3.2.3 Recombinant Protein Overexpression and Purification ... - 48 -
3.2.3.1 General Aspects of Protein Overexpression ... - 48 -
3.2.3.2 Generation of Overexpression Constructs ... - 48 -
3.2.3.3 Baculovirus Recombination ... - 49 -
3.2.3.4 Baculovirus Titer Amplification ... - 49 -
3.2.3.5 Baculovirus Titer Determination ... - 49 -
XI
3.2.3.6 Test Expression of Overexpressed Recombinant Proteins ... - 51 -
3.2.3.7 Overexpression of Recombinant Proteins ... - 51 -
3.2.3.8 Purification of Recombinant PARP-1 ... - 51 -
3.2.3.9 Purification of Recombinant His-XPA ... - 53 -
3.2.3.10 Purification of Recombinant His-XPA Fragments ... - 55 -
3.2.3.11 Determination of Protein Concentration (BCA Assay) ... - 57 -
3.2.4 Biochemistry ... - 57 -
3.2.4.1 Oligonucleotide Annealing ... - 57 -
3.2.4.2 SDS-PAGE and Protein Detection ... - 58 -
3.2.4.2.1 SDS-PAGE ... - 58 -
3.2.4.2.2 Coomassie Staining ... - 59 -
3.2.4.2.3 Western Blotting ... - 59 -
3.2.4.3 In-Vitro PAR Synthesis, PAR Purification and Size-Fractionation ... - 60 -
3.2.4.3.1 In-Vitro PAR Synthesis ... - 60 -
3.2.4.3.2 HPLC-Based PAR Size-Fractionation ... - 61 -
3.2.4.3.3 Sequencing Gel ... - 61 -
3.2.5 Functional Assays ... - 62 -
3.2.5.1 DNA Damage Induction ... - 62 -
3.2.5.2 Analysis of BPDE-Dependent Direct DSB Induction ... - 63 -
3.2.5.3 BPDE-DNA Slot-Blotting ... - 63 -
3.2.5.4 Peptide Studies ... - 64 -
3.2.5.4.1 Custom Synthesized Peptides ... - 64 -
3.2.5.4.2 PepSpot Peptide Array ... - 65 -
3.2.5.5 FAR-Western PAR Overlay ... - 65 -
3.2.5.6 Electrophoretic Mobility Shift Assay (EMSA) ... - 66 -
3.2.5.7 In-Vitro PARP Activity Assays ... - 67 -
3.2.5.8 PAR Detection Using LC-MS/MS ... - 67 -
3.2.5.9 Determination of ROS Levels ... - 69 -
3.2.5.10 Determination of NAD+ Levels ... - 69 -
3.2.5.11 Niacin Supplementation ... - 70 -
3.2.5.12 Western Blot-Based Analysis ... - 71 -
3.2.5.13 Fluorescence Microscopy-Based Analysis ... - 72 -
3.2.5.13.1 Protein-eGFP Recruitment Studies ... - 72 -
3.2.5.13.2 Fluorescence Recovery after Photobleaching ... - 72 -
3.2.5.13.3 Immunofluorescence Microscopy ... - 73 -
3.2.5.14 Flow Cytometer Based Analysis ... - 77 -
XII
3.2.5.14.1 Host Cell Reactivation Assay (HCRA) ... - 77 -
3.2.5.14.2 Cell Cycle Analysis ... - 77 -
3.2.5.14.3 Cell Death Analysis (Annexin V/PI) ... - 78 -
3.2.5.15 alamarBlue Assay ... - 79 -
3.2.5.16 Clonogenic Survival Assay (CS Assay) ... - 79 -
3.2.5.17 HPRT Forward-Mutation Assay ... - 79 -
4 Results ... - 82 -
4.1 Overexpression and Purification of Recombinant Proteins ... - 82 -
4.1.1 General Aspects of Recombinant Protein Design ... - 82 -
4.1.2 XPA and XPA-Fragments ... - 83 -
4.1.2.1 Generation of Expression Constructs of XPA Fragments ... - 83 -
4.1.2.2 Recombinant Protein Test Expression ... - 84 -
4.1.2.3 Purification of Recombinant Proteins ... - 85 -
4.1.3 PARP-1 ... - 89 -
4.1.3.1 PARP-1 Overexpression ... - 89 -
4.1.3.2 PARP-1 Purification ... - 89 -
4.2 In-Vitro PAR Synthesis ... - 91 -
4.3 XPA Non-Covalently Interacts with PAR ... - 91 -
4.3.1.1 Scheme and Model of XPA ... - 91 -
4.3.1.2 XPA’s PBM is Highly Conserved among Species and Basic Amino Acids Within are Essential for PAR-Binding ... - 92 -
4.3.1.3 The XPA’s N-terminal Fragment XPA-F1 Interacts with PAR ... - 94 -
4.3.1.4 Identification and Verification of Novel PBMs in XPA and HERC2 ... - 96 -
4.4 Reciprocal Regulation of XPA and PARP-1 ... - 96 -
4.4.1 PARP-1 Activity is Essential for an Efficient NER ... - 96 -
4.4.2 XPA-PAR Interaction Inhibits Binding of XPA to DNA ... - 97 -
4.4.3 XPA Stimulates PARP-1 Activity ... - 99 -
4.4.4 PARP Inhibition Impairs Recruitment and Mobility of XPA, XPC and p53... - 100 -
4.4.4.1 Regulation of XPA’s Recruitment to Sites of Laser-Induced DNA Damage ... - 100 -
4.4.4.2 Regulation of XPC and p53 Recruitment to Sites of Laser-Induced DNA Damage ... - 103 -
4.4.5 Altered Mobility of a PAR-Binding Deficient XPA Variant ... - 104 -
4.5 PARP-1 Mediated Response to BPDE-DNA Lesions ... - 106 -
4.5.1 Verification of PARP-1 Knockout in HeLa Kyoto Cells ... - 106 -
4.5.2 Stability and Effectiveness of PARP Inhibitor Treatment ... - 107 -
4.5.3 Validation of BPDE-Induced DNA Damage ... - 108 -
XIII
4.5.4 PARP-1 Activity-Dependent Protein Level Regulation... - 111 -
4.5.5 Exposure to BPDE Triggers PAR Formation ... - 111 -
4.5.6 BPDE Exposure can Result in NAD+ Pool Depletion ... - 114 -
4.5.7 PARylation Deficiency Reduces Colony Formation after BPDE Exposure ... - 115 -
4.5.8 PARylation Deficiency Increases Reduction Potential after BPDE Exposure ... - 117 -
4.5.9 Cell Survival after Genotoxic Treatment is not Dependent on PARP Activity ... - 119 -
4.5.10 Niacin Supplementation does not Improve Cellular Survival upon BPDE Exposure ... - 120 -
4.5.11 PARP Activity has Minor Influence on the Capacity of Repair of BPDE-DNA Lesions ... - 121 -
4.5.12 PARylation Deficiency Delays Cell Cycle Progression in Response to BPDE Exposure ... - 122 -
4.5.12.1 PARP Inhibition Enhances a BPDE-Induced G2 Phase Cell Cycle Arrest ... - 122 -
4.5.12.2 PARP Inhibition Potentiates the BPDE-induced Cell Cycle Delay ... - 123 -
4.5.13 PARylation-Deficiency Fuels BPDE-Induced Replicative Stress and the Formation of DNA Double-Strand Breaks ... - 124 -
4.5.13.1 PARylation Deficient Cells Display Increased γH2AX Levels ... - 124 -
4.5.13.2 Exposure to BPDE Induces Replicative Stress ... - 125 -
4.5.13.3 BPDE–Induced DNA Double-Strand Break Formation is Enhanced in PARP-1 Deficient Cells ... - 128 -
4.5.14 Lack of PARylation Activity Increases the Mutagenic Potential of BPDE ... - 128 -
4.6 Résumé ... - 129 -
5 Discussion ... - 131 -
5.1 Biochemical Analysis ... - 131 -
5.1.1 Biochemical Prerequisites ... - 131 -
5.1.2 Biochemical Characterization of the PAR-XPA Interaction ... - 131 -
5.2 Reciprocal Regulation of XPA-PARP-1 Protein Functions ... - 134 -
5.3 Impact of PARP-1 Activity on the Cellular Response to BPDE-Induced DNA Lesions ... - 139 -
5.3.1 Conclusion and Perspectives ... - 145 -
Contributions... - 149 -
Appendix ... - 150 -
Literature ... - 153 -
Acknowledgment ... - 182 -
- 1 -
1 Introduction
1.1 The ADP-Ribosylation System
In the 1960s two independent studies were the first that dealt with the topic of ADP-ribosylation.
Chambon and colleagues identified a new adenine-containing RNA-like biopolymer in vertebrate cells, while Collier and Pappenheimer reported the need for NAD+ for the inhibitory effect of the diphtheria toxin on the synthesis of polypeptides 11,14,15. In the following years it became obvious that these two observations are two sides of the same coin. The covalent attachment of ADP-ribose to the elongation factor 2 was identified to be the mechanism responsible for this inhibition, with ADP-ribose being derived from NAD+ 16,17. Further, it was revealed that this modification can appear in two different forms, the conjugation of single ADP-ribose moieties to target substrates [mono(ADP-ribosyl)ation;
MARylation] or the further elongation to long and branched polymers [poly(ADP-ribosyl)ation;
PARylation]. It became clear that these types of modifications can be found in all lineages of life, as well as in viruses 11,16,17. To date, three evolutionary independent superfamilies, being able to catalyze ADP-ribosylation, could be identified: the ADP-ribosyltransferase (ART), the Sirtuin, and the TM1506 superfamily 11,18-21.
1.1.1 The ART-Superfamily
Of the NAD+-dependent ADP-ribosylation systems the ART superfamily is the most diverse in terms of sequence, structure, active site residues and target molecules 11. Primary diversification emerged in account of complex bacterial conflict systems, with ARTs being involved in offense and defense strategies. These include toxin-antitoxin (T-A) systems, virus-host interactions, intraspecific antagonisms, symbiont/parasite effectors/toxins and resistance to antibiotics 11. In more than 20 independent occasions eukaryotes acquired several ARTs by lateral transfer. In this, the acquisition of the family of poly(ADP-ribose)polymerases (PARPs) happened early in eukaryotic development, while others were only gained at later time points 11,22. In the course of eukaryotic evolution many ARTs retained their roles, being involved in anti-pathogen strategies. Others developed functions related to their ancestral purposes in T-A systems, now being mediators of apoptosis and cell death, while still others evolved to play roles in core regulatory systems 9,11,23-25.
Thus, it is not surprising that among the first identified protein modifications were many of those conflict systems, e.g. the mono(ADP-ribosyl)ation of bacterial toxins 17,18 or of proteins of host cells infected with the bacteriophage T4 17,26. Over time, mono- and poly(ADP-ribosyl)ation came into focus during the research of diverse eukaryotic elements, like the modification of nuclear proteins by PARPs, the cytoskeleton by MARTs [mono(ADP-ribose)transferases], or surface proteins by ECTO-MARTs.
It further became clear that especially the family of PARPs was involved in a variety of cellular aspects, as epigenetics, DNA repair, apoptosis, signaling and even complex biological processes like long-term memory formation 11,19,27,28.
The ART superfamily can be roughly divided into three higher order clades based on their active site residue configuration, the H-H-h clade, the H-Y-[EDQ] clade and the R-S-E clade (Figure 1.1) 11. Herein, the H-Y-[EDQ] clade underwent a substantial diversification based on prokaryotic conflict systems. In one of its subclades the family of PARPs (discussed in detail in the following section) evolved as effectors of such inter-organismal conflicts 29. This group compromises on the one hand, the polymer-forming PARPs (PARP1/2 and PARP5/6) and on the other hand mono(ADP-ribosyl)ating and catalytically inactive PARPs (PARP3/4 and PARP7-17) 19,22,30.
- 2 -
Figure 1.1: Reconstructed evolutionary history of the ART superfamily. The ART superfamily developed in prokaryotic systems in account of several complex conflict systems. Primary diversification evolved early on and gave rise to three major clades: the H-H-h and H-Y-[EDQ] clade, comprising the ARTD family members, and the R-S-E clade, containing the ARTC family members. Eucaryotes acuired these ART systems in several independent occasions by lateral transfer. Herein, the accuisition of the PARP family (H-Y-[EDQ]) happened early in eucaryotic development, homologous of PARP-1 beeing present in the last comon eukaryotic ancester. The structure beyond the tree depicts the idealized topology of the common ART fold. Adapted from 11.
- 3 -
1.2 The PARP Superfamily
1.2.1 Classification and Organisation of PARPs 1.2.1.1 Evolutionary Clustering
The family of poly(ADP-ribose)polymerases (PARPs) carries the evolutionary conserved amino acid triad H-Y-E and configurations derived thereof in their catalytic center. This family most likely evolved in all eukaryotic organisms, but was later independently lost in some lineages. To date, in five of six eukaryotic supergroups PARPs have been identified, but seem to be lost in many fungi species, including the model systems saccharomyces cerevisiae and schizosaccharomyces pombe 22,31.
Recently, in the attempt to simplify as well as specify the organization of these proteins a new nomenclature was proposed by Hottiger et al. for the families of H-Y-E and R-S-E ARTs. Due to their structural homology to diphtheria toxin or cholera toxin, it was suggested to place these proteins in the groups of ADP-ribsoyltransferase diphtheria toxin-like (ARTD; H-Y-E class) and ADP- ribosyltransferase cholera toxin-like (ARTC; R-S-E class). While ARTC proteins cover the group of ECTO-MARTs, the PARP family is resembled by the ARTDs 9.
The catalytic core fold of the ART superfamily consist of two distinct units of β-sheets, each composed of three antiparallel strands (strand order 4-5-2//1-3-6), flanked by α-helices. Strand 1 (H), strand 2 (Y) and strand 5 (E) herein comprise the catalytic triad 11,29,32-34.
Figure 1.2: Schematic comparison of the domain architecture of the human PARP (ARTD) family. While the catalytic ART domain can be found in all members of the PARP family the composition of the remaining domain architecture varies greatly between the family members, according to their specific functions. Adapted from 9.
- 4 -
At least two PARP family members are evolutionary old and could likely be found in the last eukaryotic common ancestor (LECA), one of them showing a strong similarity to the human PARP-1 protein, likely already being involved in DNA damage responses 22. In the course of eukaryotic evolution, further diversification to distinct lineages occurred (e.g. tankyrases and vPARP). This was further accompanied by fusion to several distinct domains essential for their specific roles 9,11.
1.2.1.2 The PARP Family
The human PARP family consists of 18 members, unified by their NAD+-binding pocket including the strongly conserved PARP signature motive. This motive is formed by a beta-alpha-loop-beta-alpha structure, composed of strands and helices 3 and 4, inter-connected by the D-loop, of PARPs catalytic center. While this structure is highly conserved and can be found in all members of the PARP family, other protein domains are strongly diverse and depend on the specific protein functions (Figure 1.2)
5,9,34-36.
1.2.1.3 Structure of PARP-1
The founding member of the PARP family, PARP-1, is the best studied and characterized of all 18 PARP proteins. Cloning of the PARP-1 cDNA in the late 1980s by three independent groups 37-39, purification, biochemical analysis and finally resolution of the PARP-1 crystal structure facilitated this characterization 34,40,41.
PARP-1 (EC 2.4.2.30) is a 113 kDa protein, constitutively expressed with a highly conserved modular architecture. It consists of three major domains. The N-terminal DNA-binding domain is composed of two structurally homologous zinc fingers, Zn1 and Zn2 (PARP-like zinc fingers), which are apt to detect special DNA structures rather than specific DNA sequences 42,43. Separated by a caspase cleavage site and PARP-1’s nuclear localization sequence (NLS) follows a third, structurally different zinc finger (Zn3), which is supposed to be less important for DNA-binding but relevant for inter-domain communication 5,44,45. The automodification domain includes a BRCT (BRCA1 C-terminal) domain, a protein-protein interaction fold found in many DNA damage response and cell cycle control proteins.
Further, this area covers multiple glutamate residues on which PARP-1 is automodified upon activation
46,47. The catalytic domain (CAT) contains three subdomains, the WGR, the HD and ART domain. The WGR domain is named after a highly conserved Trp-Gly-Arg amino acid motif, which is supposed be important for DNA-binding, thus being essential for the DNA-dependent activation of the protein 40,46. The six α-helices forming the up-up-down-up-down-down motif of the HD (helical domain) have a regulative, autoinhibitory function. In the absence of a PARP-1 stimulus, a portion of this domain blocks efficient NAD+-binding to PARP’s catalytic center. Upon strand break-binding, this region unfolds and Figure 1.3: Schematic presentation of the human PARP-1 architecture. PARP-1 is composed of three major regions, the DNA-binding domain, the automodifcation domain and the catalytic domain. These domains can be further subdivided into smaller units, the two homologous zinc fingers Zn1 and Zn2, the structurally different zinc finger Zn3, the BRCT (BRCA C-terminal) domain, the WGR (tryptophan-glycine-arginine) domain, the helical (HD) domain and the ART (ADP-ribosyltransferase) domain. The nuclear localization sequence (NLS) and caspase cleavage site are located between Zn2 and Zn3. Adapted from 5.
- 5 -
thus transmits the activation signal to the ART domain by enabling NAD+-binding 48. The ART domain is responsible for the actual catalytic activity. Its ART and PARP signature motif form a pocket for NAD+-binding, catalysis and ADP-ribose transfer to target amino acid residues. While Zn1, Zn3, WGR and the CAT domain are essential for DNA-dependent PARylation, Zn2 and the automodification domain seem to be dispensable for strand break-triggered enzyme activity 5,40,45-47,49. Despite not being essential, DNA-binding by Zn2 is believed to be important for efficient response of PARP-1 to DNA double-strand breaks (DSBs), but not to single-strand breaks (SSBs) 40,46,49,50.
1.2.1.4 MARylation and PARylation Activities of PARP Family Members
To date four proteins have been characterized in humans with bona fide polymer formation abilities:
PARP-1 51, PARP-2 52,53, tankyrase 1 (PARP-5a) 54,55 and tankyrase 2 (PARP-5b) 56,57. For the remaining fourteen members of the PARP family either only mono(ADP-ribosyl)ation was shown or postulated (PARP-3/4, PARP-6-8, PARP-10-12, PARP-14-16, TRPT1) or they are believed to be catalytically inactive (PARP-9, PARP-13) 9,58-61.
1.2.2 PAR Metabolism
While polymer formation has been described for four members of the PARP family, a DNA-dependent stimulation of enzyme activity was only shown for PARP-1/2 (PARylation) and PARP-3 (MARylation)
52,62-65. PARP-1 is responsible for the vast majority of cellular PAR formation (~85%), with PARP-2 being involved in the synthesis of the remaining ~15%. The overall impact of the other polymer forming PARPs is believed to be rather limited 60,65. Even in the absence of DNA damage a basal PARP-1 enzyme activity can be detected. Upon exposure to genotoxins, like alkylating and oxidizing agents, ionizing radiation and non-ionizing UV-irradiation, PAR formation can strongly increase 66.
Apart from DNA-dependent stimulation of enzyme activity, polymer formation by PARP-1 is known to be triggered or modulated by several DNA damage-independent mechanisms. PARP-1 binding to specific, undamaged DNA motifs and nucleosomes 67,68, posttranslational modification of PARP-1 by phosphorylation and acetylation 69,70 and direct protein-protein interaction (Ying Yang 1, pERK2) 71,72 have been reported to be involved in the stimulation of PARylation activity. Further, mono(ADP- ribosyl)ation of PARP-1 by SIRT6 and probably PARP-3 was suggested to serve as ‘kickstart’ for PARP-1 automodification 73,74. Nevertheless, probably best understood are the mechanisms induced by DNA strand interruptions, leading to the activation of PARP-1. PAR catalysis upon strand break detection is rapid, appears after seconds and is critical for subsequent damage signaling and the tightly organized repair of lesions by the DNA repair machinery.
1.2.2.1 PAR Anabolism
Mechanistic Aspects of PARylation
The DNA-binding domain of PARP-1 shows a strong affinity for DNA strand interruptions. The occurrence of hydrophobic interactions of the enzyme’s zinc fingers with exposed bases of DNA single- and double-strand breaks leads to conformational changes and the consequent enzymatic stimulation of PARP-1 40. The question if for strand break detection and subsequent catalytic activation of PARP-1 protein-dimerization is required is controversially discussed 40,43,49,75-79.
In a free, unbound condition the PARP-1 protein was described as an extended monomer. Upon DNA- binding conformational changes occur, leading to protein compaction and reorganization of protein domains 40. Based on crystal structure analysis of the individual subdomains in complex with blunt-end duplex DNA, Langelier et al. proposed a model of monomeric PARP-1 DNA-binding and subsequent catalytic activation 40. The structural homologous Zn1 and Zn2 interact primarily with the DNA by the
- 6 -
use of two structural features: The ‘backbone grip’ facilitates the interaction with the uninterrupted segment of the DNA phosphate backbone (sequence unspecific), while the ‘base stacking loop’ engages the exposed terminal base pair via hydrophobic interaction. This structural DNA feature of an exposed hydrophobic face can be found in several DNA structures bound by PARP-1, in single- and double- strand breaks, as well as in abnormal, but continuous structures like DNA hairpins and cruciform DNA
80. The ‘base stacking loop’ embodies a flexible element, only requiring the common feature of hydrophobic interactions with exposed bases, by which it enables PARP-1 sensing various types of DNA aberrations 49. Upon PARP-1/DNA complex formation, Zn1 and Zn3 transfer an activating signal to the WGR domain via the ‘base stacking loop’ and an ‘extended loop’ of Zn3. This causes a destabilization of the HD, the autoinhibitory part of the CAT domain 40,48. The relevance of this region is highlighted by several mutation studies (L698, L701, L713), which resulted in DNA-independent PARP-1 hyperactivation 40,81. Of note, the degree of HD unfolding correlates with catalytic activation and thus the catalytic output. The magnitude of activity depends on the type of DNA damage detected by PARP-1 (e.g. blunt end vs. base overhang) 42,62,82. It is tempting to speculate that varying DNA damages alter the position of Zn1’s ‘base stacking loop’ and thus control the level of PARP-1 activation
49. Further, it can be assumed that other modes of PARP-1 activation (protein-protein interaction, posttranslational modification) also operate by direct destabilization of the HD or by using the mediating WGR domain 48.
Ali et al., on the other hand, proposed a model in which both zinc fingers work as a functional unit and PARP-1 dimerizes in order to become fully active (trans-activation). In this model, the homodimer binds both ends of the double-strand break, keeping them close for subsequent DNA repair 78.
Figure 1.4: Model of step-wise assembly of PARP-1 monomer at the sites of DNA single-strand breaks.
Binding of Zn2 on the 3’ DNA stem initiates further sequental assembly. Eventually this causes activation of the ART domain by local unfolding of the inhibitory HD domain. Adapted from 8.
- 7 -
Finally, Eustermann et al. used NMR spectroscopy to describe the dynamics of PARP-1 domain organization at the sites of DNA single-strand breaks 8. PARP-1 binds to DNA SSBs as a monomer, with a zinc finger orientation similar to what was described by Ali et al., Zn2 binding the 3’ and Zn1 the 5’ stem of the DNA. This results in a strong bending of the DNA and triggers the further stepwise self-assembly of the PARP-1 domains, ultimately leading to catalytic activation and cis automodification (Figure 1.4).
Biochemical Aspects of PARylation
PARP-1 is involved in numerous cellular processes and has been reported to interact with several different DNA structures, independent of their sequence. It is conceivable that to be able to recognize this diversity of activating structures and at the same time preserve the enzymes’ specificity, several modes of PARP-1 activation are possible, probably resulting in varying degrees of catalytic activity.
Anyhow, upon activation PARP-1 consumes NAD+, nicotinamide is released and the ADP-ribose unit is covalently attached to PARP-1 itself or other target proteins. Subsequently, this mono(ADP-ribose) unit can be further elongated to form polymer chains of up to 200 moieties 17,19. An additional level of complexity is added by occasional branching every 20-50 ADP-ribose units 83-86.
Figure 1.5: Metabolism of mono- and poly(ADP-ribosyl)ation. PARPs consume NAD+, release nicotinamide and covalently attach ADP-ribose to target proteins. MARylation can be reversed by the activity of MacroD1, MacroD2, TARG1 and ARH1 (not depicted). The initial ADP-ribose unit can be further elongated by some members of the PARP family. PARG has endo- and exoglycolytic functions, and is involved the removal of PAR chains from target proteins, as is ARH3 (not depicted). Finally, TARG1 can detach intact PAR chains form proteins, releasing uncleaved, long and branched polymer. Adapted from 13.
- 8 -
PARP-1’s catalytic activity is triggered by the destabilization and removal of the autoinhibitory HD subdomain from its catalytic center 48. This permits the binding of NAD+ to PARP-1’s nicotinamide binding pocket, mediated by the histidine side residue of the H-Y-E catalytic triad 61,87,88. The glutamate residue catalyzes a substitution reaction by forming an oxocarbenium ion as an intermediate, presented for nucleophilic attack by the acceptor nucleophile at the 1’’ position of the ribose unit 85,89. This initial mono(ADP-ribose) moiety can subsequently be elongated by gradually adding ADP-ribose units via 2’-1’’ o-glycosidic ribose-ribose bonds. The glutamate residue was also linked to the occasional branching via a 2’’-1’’’ ribose-ribose o-glycosidic linkage 41,86,90.
Amino acid side chains for initial ADP-ribose linkage to target proteins have been identified as predominantly glutamate, aspartate (γ and β carboxyl group, respectively) and lysine residues (ε-amino group) 46,47,61,91-94. The glutamate in the catalytic triad plays a substantial role in mediating the transfer of ADP-ribose units. But while it is not essential for the initial ester bond in mono(ADP-ribosyl)ation, here the carboxylate group of glutamate and aspartate are sufficient to mediate the initial monoADP- ribose transfer 59,85,86, it is substantial for chain elongation and branching. However, mutation studies revealed that the presence of glutamate at this catalytic site is less relevant rather than the presence of an acidic side chain. E988D reduced the polymer forming ability about 20-fold compared to a 2,800- fold reduction observed in E988Q/K mutants 85. The presence of glutamate at this site was formerly used to predict polymer formation abilities of the PARP family members. Continuative investigations indicate that the triad is not the sole determinant of polymer forming ability 61. Several bacterial toxins, carrying a glutamate at this active site, have been proven to only mono(ADP-ribosyl)ate, and on the other hand a E988K mutant showed a strongly reduced, but still at least oligo(ADP-ribosyl)ation activity. Further investigations are necessary to identify the detailed structural features and mechanisms needed for polymer formation 5,59,95.
Functional Aspects of PARylation
In response to DNA damage the basal cellular PAR levels are rapidly increased up to 500-fold. PAR chains are covalently attached to target proteins, of which PARP-1 itself is the major target 96. As a result of this, three major consequences can be outlined in response to DNA damage. Automodification of PARP-1 as well as heteromodification of surrounding histones establishes a chromatin-bound platform at the site of DNA damage, used for the chromatin remodeling necessary for DNA repair.
Second, this provides the basis for efficient recruitment of downstream repair factors and the spatio- temporal assembly of the relevant DNA repair machineries. Finally, the DNA damage load is sensed and signaled by the extent of PAR formation. Depending on the magnitude of polymer formation either DNA repair is facilitated (low-medium DNA damage) or cell death is triggered (extensive DNA damage) 97.
Besides its direct involvement in DNA repair, PARylation plays numerous roles in DNA damage related and unrelated cellular aspects. Increasing numbers of covalently PAR-modified proteins have been identified in the last decades 98. The addition of the highly negatively charged PAR chains to target proteins can have a dramatic impact on protein functionality by altering structural conformation, steric hindrance, influencing protein-protein or protein-nucleic acid interactions. This includes the enzymatic activity of PARP-1 itself. Upon automodification PARP-1’s catalytic activity wanes progressively. This can be ascribed to a loss of affinity to DNA, which is dramatically reduced due to electrostatic repulsion by the negatively charged polymer, and probably conformational reorganization of the PARP-1 protein
99-102. This is highlighted by the observation of cytotoxic PARP-1 trapping as protein-DNA complexes at the sites of DNA damage when its catalytic activity is inhibited 100,103-106. Such and other influences of PARylation can be observed in many proteins and thus it is not surprising to see a significant
- 9 -
influence of PARP-1 activity on several cellular processes, such as telomere homeostasis, cell cycle regulation, spindle pole function, oncogene-related signaling, protein stability, transcriptional regulation, differentiation, cell death, hypoxic response and inflammation 31,83,97,107-109.
1.2.2.2 PAR Catabolism
The wide range of PARP-dependent processes makes it conceivable that PAR formation needs to be tightly controlled in a spatio-temporal manner. Indeed, depending on the type of DNA damage, polymer synthesis can often be observed already seconds after damage induction. PAR levels climax after 4-5 min and thereafter rapidly return back to basal levels. The swift turnover (t1/2= 1-6 min 110) is mediated by a set of enzymes, which are able to degrade the polymer to oligo- and mono(ADP-ribose) and remove the terminal ADP-ribose moiety from target proteins. Unbound mono(ADP-ribose) is finally catabolized to AMP and ribose 5-phosphate 83. Polymer degrading capabilities have been identified in two structurally unrelated proteins, the macrodomain containing poly(ADP-ribose)glycohydrolase (PARG) and the ADP-ribosylhydrolase 3 (ARH3) 5. Subsequent removal of mono(ADP-ribose) moieties from proteins is performed by the macrodomain containing proteins TARG1, MacroD1, MacroD2 or the ARH1 protein 13.
PARG is able to degrade PAR in a timescale of minutes, with a higher efficiency for linear PAR rather than branched polymer 110-113. The relevance of this protein for the cell and hence organisms is emphasized by the observation of embryonic lethality of PARG knockout mice 114. Five isoforms of PARG have been identified, located in different cellular compartments. Of these, the full-length PARG (110 kDa) is the only one found in the nucleus 115-118. PARG is capable of binding and o-glycosidic cleavage of PAR by means of its macrodomain. As distinguished from other macrodomain-containing proteins, PARG’s catalytic center contains a catalytic loop bearing the PARG signature motif (GGG- X6-8QEE) 119-122. This structural feature enables PARG to be active as endoglycohydrolase. However, PARG preferentially binds to and sequentially degrades terminal ADP-ribose moieties 111,122-125. It was suggested that PARG’s endoglycolytic activity only becomes of relevance in case of increasing PAR/PARG ratios, situations of high DNA damage load and subsequent PAR-dependent triggering of cell death 124,126. While PARG is capable to be active as an endoglycohydrolase, sterical hindrances prevent the cleavage of branching points as well as the removal of the terminal ADP-ribose moiety from proteins 121,124,127.
Another macrodomain containing protein, the terminal ADP-ribose glycohydrolase 1 (TARG1), has been shown to hydrolyze the mono(ADP-ribose) linkage to acidic side residues 93,128. Further, TARG1 is able to cleave the ester linkage between proteins and intact PAR chains, releasing full-length polymer, but cannot further hydrolyze free PAR 93. This feature is believed to be essential for Parthanatos, a mode of cell death triggered by free PAR chain-mediated AIF release from mitochondria upon excitotoxicity in neural cells, or massive DNA damage in other cell types 129,130.
In mammals three ARHs are described 28. Of these, ARH1 is capable to hydrolase MAR from arginine
131-133. ARH3, even if less active than PARG, is involved in the hydrolysis of polymer 132,133. It was suggested that the function of ARH3 might be in the degradation of protein-free PAR 134 or being the primary PAR-degrading enzyme of the mitochondrion 117,132,133,135. The role of ARH2 remains elusive.
MacroD1 and MacroD2 are macrodomain proteins, able to cleave the bond between the terminal MAR and the protein 136,137. Both proteins, as well as TARG1 remove mono(ADP-ribose) exclusively from acidic residues and do not act on lysines 128,138.
- 10 -
TARG1, MacroD1, MacroD2 and ARH3 further have the capacity of cleavage of o-acetyl-ADP-ribose (OAADPR), the side-product of SIRT1-mediated protein deacetylation 136,139,140.
1.2.3 Non-Covalent PAR-Binding Modules
Catalytic activation of PARP-1 triggers massive local PAR synthesis. Covalent attachment of PAR to target proteins alters physico-chemical properties of the modified proteins. Early on it became clear that not all of the PAR-dependent observations could be contributed to covalent PARylation 141-143. Non- covalent interactions between attached or free polymer and increasing numbers of PAR-binding proteins added another level of complexity to the world of PARylation 144-146. Up to know, six specialized PAR-binding modules have been identified, each recognizing different structural features of the PAR chain 69.
The PAR-Binding-Motif
Of all PAR recognition modules the PAR-binding motif (PBM) is the most abundant one and was the first discovered module able to interact non-covalently with poly(ADP-ribose). Many proteins involved in DNA damage response and repair have been identified to contain a PBM, suggesting that PARylation at the site of DNA damage provides a platform to recruit DNA repair proteins 147. Multiple sequence alignments resulted in the initial determination of the motif. A loose pattern of alternating hydrophobic and basic amino acids (HxBxHHBBHHB) downstream of an arginine/lysine rich region was identified
147-149. The PBM displays only a limited degree of consent and many in-silico identified PBMs have been disproven to bind PAR. Thus, it was suggested that PAR-binding is more dependent on conserved physical and structural properties rather than on a fixed sequence 60,150. MS-based screens identified numerous proteins, interacting non-covalently with polymer via this motif and led to a refinement of the proposed PBM consensus sequence 151. Of note, in some proteins a selective affinity to long and branched polymer has been observed (e.g. XPA, DEK), while others seemed to be less specific (e.g.
p53) 152,153. Although little is known on the mechanistic level how PAR interacts with the PBM and influences protein functions, many PBMs do overlap with important regulatory domains 147. It is conceivable that steric hindrance, electrostatic repulsion and distortion of critical protein domains might be responsible for the functional consequences induced by PBM-PAR interactions, such as destabilization of protein-protein or protein-ligand interactions 152-155. By this means, the PBM is able to contribute to the recruitment of DNA damage response factors to sites of PARylation, the stabilization or destabilization of multi-protein complexes and the alteration of their functionality 60. The GAR and RRM motifs
Recently, two, so far poorly characterized, alternative PBMs have been described, the glycine and arginine rich domain (GAR) and the RNA recognition motif (RRM) 156-159. Due to the high abundance of positively charged arginine residues, the GAR offers a suitable binding surface for the negative polymer 60. GAR-containing proteins have been primarily identified to be involved in RNA metabolism and chromatin processing 157-159. The RRM on the other hand is associated with RNA- and single-strand DNA-binding proteins 156.-
The PAR-Binding Zinc Finger
To date, the PAR-binding zinc finger (PBZ) was only detected in three human proteins (APLF, CHFR and DCLRE1A) 160. Interestingly, this module links PARylation with another posttranslational modification, since CHFR has an E3 ubiquitin ligase activity with functions in the antephase
- 11 -
checkpoint. Dependent on the automodification of PARP-1, CHFR is able to trigger the delay of mitotic entry by polyubiquitination/degradation of PARP-1 in response to mitotic stress 160-164.
The WWE Domain
The crosstalk between PARylation and ubiquitination is further emphasized by proteins carrying the WWE domain. This module is named after the conserved amino acids tryptophan and glutamate, flanked by regions with a low degree of sequence conservation 60,165. Of the 12 human proteins with such a module, six proteins are associated with ubiquitination, while most of the remaining belong to the PARP family 165. A direct connection between the two posttranslational modifications could be revealed in the regulation of the Wnt/β-catenin signaling pathway by Iduna. Here, covalent PARylation of axin is essential for the recognition and subsequent ubiquitination/degradation by Iduna 165,166. The Macrodomain Fold
The macrodomain fold is a globular domain, named after the histone variant macroH2A, in which it was first described 167. It was identified in all kingdoms of live, as well as in a series of viruses where it might be involved in ADP-ribose related T-A systems 168. Within the PAR-recognition modules the macrodomain fold is unique in respect to its dual function of binding and processing mono- and poly(ADP-ribose) in some proteins (PARG, TARG1, macroD1 and macroD2).
1.2.4 Biological Functions of PARP-1 Chromatin Structure and Organisation
The higher-order structure and density of chromatin compaction has determining influence on the accessibility of DNA-associated factors, thus controlling repair and transcription. The degree of chromatin packing is defined by the composition of factors associated with the DNA (histones and non- histones). As one of the earliest sensors of DNA damage and mediator of DNA repair, PARP-1 is involved in the modulation of chromatin packing by three distinct modes 169.
First, PARP-1 covalently and non-covalently modifies chromatin-associated factors, such as histones, high-mobility group proteins (HMG), heterochromatin protein 1 (HP1) and macroH2A 92,170-172. Modification with the negative polymer may alter the proteins affinity to DNA and remove them from the site of PARylation. By doing so, PARP-1 activity directly controls chromatin architecture, and grants or denies chromatin accesses for the DNA repair machinery or transcription factors 173-175. Second, PARP-1 competes with the histone linker H1 for binding to nucleosomes. PARP-1 binding to nucleosomes results in local compaction of the chromatin and transcriptional repression. Upon PARP-1 activation, the protein automodifies itself and dissociates from the chromatin. This results in chromatin decondensation and restoration of transcription 67,176,177.
Third, PARP-1 recruits chromatin remodelers, such as ALC1 or NURD to the sites of PARylation.
Here, these factors deploy their nucleosome sliding activity, thus facilitating the binding and activity of DNA repair factors 178-180.
Transcriptional Regulation
Through its role in chromatin regulation, PARP-1 is involved in gene transcription, granting and denying excess of transcription factors by altering compaction status. Beside this, a direct impact on several central transcription factors is known 181.
- 12 -
The tumor suppressor p53 plays a key role in the cellular response to genotoxic stress. The decision if a cell undergoes DNA repair, cell cycle arrest, senescence or cell death is mainly mediated by p53 182. PARP-1 and p53 have been shown to interact in-vitro and in-vivo 183-185. p53 was reported to be covalently PARylated and interacts with PAR via one of its PBMs 147,186,187. Non-covalent interaction between PAR and p53, influences p53’s DNA-binding affinity and is believed to be involved in nuclear localization and regulation of p53’s transcriptional activity 154,188. PARP-1 further modulates inflammation and is essential for the transcription of proinflammatory genes, such as TNFα, by binding to NF-κB. This PARylation-independent interaction increases NF-κB’s DNA-binding affinity and transcriptional activity. Translocation of NF-κB from its cytoplasmic detention was further shown to be PARP-activity dependent 189. A negative regulation was shown for the TATA-binding protein (TBP).
PARylation of this protein blocks the formation of the preinitiation complex (PIC) necessary for general transcription start 190,191.
Besides this direct influence on transcription, PARP-1 is implicated in DNMT1 (DNA (cytosine-5)- methyltransferase 1)-mediated DNA methylation. PARP-1 forms a complex with CTCF (CCCTC- binding factor) and DNMT1 and modifies both proteins either covalently or non-covalently with PAR polymer 192,193. PARP inhibition causes a DNMT1-dependent hypermethylation of DNA, while PARP activity is associated with hypomethylation, thus influencing epigenetic transcriptional regulation 194-
197.
Protein Stability and Turnover
The observation that one of the non-covalent PAR-binding modules, the WWE domain, is predominantly found in E3-ubiquitin ligases hints for a tight cross-talk between PARylation and ubiquitination 60. Indeed, a PAR-dependent ubiquitination and subsequent proteasomal degradation was revealed in the context of Wnt signaling. The E3-ubiquitin ligase Iduna/RNF146 is known to bind and become activated by both, short chains of polymer (transferred by tankyrases) as well as long polymer chains (PARP-1). Upon PARylation of axin by tankyrases, Iduna/RNF146 binds via its WWE domain and marks the protein for degradation by ubiquitination. As a result β-catenin-dependent transcription takes place 126,166,198,199. Besides this pathway, Iduna/RNF146 is implicated in the modification of a number of DNA repair factors, such as PARP-1 and 2, XRCC1, Ku70/80 and DNA ligase III and also herein its activity is PAR-dependent 166.
The majority of patients suffering of cherubism carry a mutation in the protein 3BP2, thus disturbing the interaction with tankyrase 2. Without the PARylation by tankyrase 2 this protein evades ubiquitination by Iduna/RNF146 and subsequent proteasomal degradation. This ultimately leads to systemic inflammation and the cherubism phenotype 200,201.
NAD+ Metabolism
NAD+ is a molecule of vital importance, placed in the midst of several fundamental cellular processes.
The relevance of this molecule is highlighted by the vitamin deficiency disease pellagra (disease of the four Ds; dermatitis, diarrhea, dementia, death). Pellagra is caused by malnutrition in form of lacking intake of niacin (vitamin B3, nicotinic acid) and tryptophan, precursors for NAD+ synthesis 202.
NAD+ is probably best known for its role as a coenzyme for several reduction-oxidation reactions.
NAD+ and its reduced form NADH serve as key intermediates for energy transfer from different metabolic pathways and link the fundamental catabolic pathways of glycolysis and Krebs cycle (TCA) to the electron-transfer chain and oxidative phosphorylation (ATP synthesis). In these redox reactions NAD+ and NADH are interconverted but not consumed 10.
- 13 -
However, NAD+ serves as a cofactor in several other cellular processes in which the molecule is consumed. In each of these processes NAD+ is hydrolyzed, nicotinamide is released and the free energy is used to drive the ensuing reaction with ADP-ribose 19. CD38 and CD157 consume NAD+ in order to synthesize cyclic ADP-ribose for calcium signaling 203. Enzymes of the ART and PARP families transfer ADP-ribose moieties to target substrates, further elongated to long chains by some PARP members. Sirtuins utilize NAD+ for deacetylation of proteins, forming the by-product o-acetyl-ADP- ribose.
PARylation as a result of genotoxic stress is probably the major challenger of NAD+ pools, transiently reducing concentrations up to 80 % within minutes. Although this only depicts a considerable degree of DNA damage and at more moderate challenges, only 5-10 % of the cellular NAD+ is depleted, it is conceivable that mild but continuous PARylation still can jeopardize the cellular machinery 19.
To maintain NAD+ concentrations despite the continuous depletion by NAD+-consuming enzymes, several synthesis pathways are available with different NAD+ precursors.
The major dietary source of precursors for NAD+ synthesis is nicotinic acid. This molecule can be transformed to NAD+ in three steps via the Preiss-Handler pathway. De-novo synthesis is performed from tryptophan, the pathway merging at a point with the Preiss-Handler pathway. The salvage pathway is probably the most important one in maintaining cellular NAD+ levels. It recycles the byproduct nicotinamide to form NAD+ and herby conserves the energetic potential but also relieves the cell from the inhibitory influence which nicotinamide has on NAD+-consuming enzymes 10,204.
NAD+ levels and NAD+/NADH ratios have been reported to be important elements and determine life and health span of yeast, worms and mammalian cells 205-208. These observations are tightly linked to the protein family of sirtuins. SIRT1 and SIRT2 have been shown to positively affect aging in mice in a NAD+-dependent manner
209,210. Although both are involved in cellular homeostasis and wellbeing, sirtuins and PARPs do show antagonizing influences to a certain extent. Both compete for NAD+ with comparable Km values (PARP- 1<SIRT1) and upon PARP-1 stimulation SIRT1 activity was reported to be reduced 10. On the other hand, PARP-1 is inhibited by SIRT1 via direct deacetylation as well as by transcriptional regulation 211,212. The severe consequences of dysregulation of the PARP-1 and SIRT1 NAD+ metabolism are shown by a series of recent studies. The deficiency of the DNA repair factor XPA causes the clinical phenotype of Xeroderma Pigmentosum. This disease is linked to increased disposition of cancer and neurological degeneration. Together with other neurodegenerative disorders (CS and AT) caused by defects in
Figure 1.6: NAD+ metabolism in different cellular compartments.
Several different NAD+ precursors can be taken up by the cell and metabolized by different pathways to NAD+. This is on the one hand an essential coenzyme for energy metabolism and redox systems and links glycolysis and Krebs cycle to the respiratory chain. On the other hand it is consumed by central key enzymes, such as ARTs, PARPs and sirtuins and thus needs to be resynthesized constantly. Adapted from 10.
