Research Collection. The role of Poly(ADP-ribose) formation in DNA excision repair in Xenopus egg cell-free lysates. Doctoral Thesis.
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1 Research Collection Doctoral Thesis The role of Poly(ADP-ribose) formation in DNA excision repair in Xenopus egg cell-free lysates Author(s): Höfferer, Liane Publication Date: 1994 Permanent Link: Rights / License: In Copyright - Non-Commercial Use Permitted This page was generated automatically upon download from the ETH Zurich Research Collection. For more information please consult the Terms of use. ETH Library
2 DISS. ETH NO The Role of Poly(ADP-Ribose) Formation in DNA Excision Repair in Xenopus Egg Cell-Free Lysates A Dissertation submitted to the SWISS FEDERAL INSTITUTE OF TECHNOLOGY for the Degree of DOCTOR OF NATURAL SCIENCES Presented by LIANE HOFFERER Mag. Rer. Nat. Born September 26th, 1960 Citizen of Puchenau/Linz, Austria Accepted on the recommendation of PROF. DR. KASPAR H. WINTERHALTER, EXAMINER PROF. DR. FELIX R. ALTHAUS, COEXAMINER 1994
3 6 SUMMARY
4 Poly(ADP-ribose) polymerase (PARP) is a versatile enzyme in the cell nucleus catalizing a highly energy consuming process. Experimental evidence, based mainly on inhibitor studies, suggested that poly(adp-ribose) turnover has an essential function in DNA excision repair. To address the role of poly(adpribosyl)ation in DNA repair directly, a Xenopus laevis egg cell-free system was established which is able to repair i) pyrimidine dimers, (6-4) photoproducts, and AAF adducts by nucleotide excision repair (NER), and ii) pyrimidine hydrates, alkylated bases and thymine glycols by base excision repair (BER) with high efficiency. The main findings were that repair occurs via a distributive mode and that the poly(adp-ribosyl)ation system is differentially involved in the two excision repair pathways investigated. During the repair of OsO4-and MNNGdamages which are substrates for BER, poly(adp-ribose) polymers were syn thesized and prevention of polymer production led to a drastic inhibition of repair. Contrastingly, the repair of UV- and AAF-damage did not activate PARP and the presence of the competitive inhibitor benzamide did not affect NER. These data suggest that poly(adp-ribosyl)ation is directly involved in BER but apparently not employed in NER in the Xenopus egg lysate system. However, the involvment of PARP in BER appears to be a negative one, in that the enzyme binds tightly to DNA strand breaks and competes with repair enzymes for access to the damaged site. Only upon automodification, PARP is released from DNA thus allowing access of repair enzymes. Xenopus lysates with inactivated PARP exhibit full DNA excision repair in the absence of poly(adp-ribose) turnover. So, if PARP is dispensable in DNA excision repair, what is it doing then? Data emerging from different groups including ours led to a new sight of poly(adpribose) function. It might have a role in the prevention of illegitimate recombi nation processes and in sensoring DNA damage. General agreement exists in the view that poly-(adp-ribose) has a role in the maintenance of genome integrity and chromatin metabolism. 115
5 7 ZUSAMMENFASSUNG
6 Die Poly(ADP-ribose) polymerase (PARP) ist ein vielseitiges Enzym des Zellkerns, welches einen energetisch aufwendigen Mechanismus katalysiert. Aufgrund experimenteller Daten, die vorwiegend auf Enzym-Hemmstudien basieren, wurde vermutet, dafi Poly(ADP-ribose) eine essentielle Funktion bei der DNS Exzisions-Reparatur tragt. Um die Funktion der Poly(ADP-ribosyl)ierung bei der DNS Reparatur zu studieren, wurde ein zellfreies Lysat aus Xenoptis laevis Eiern hergestellt. Dieses konnte i) Pyrimidin-Dimere, (6-4) Photoschaden und AAF-Addukte iiber den Nukleotid-Exzisions-Reparatur (NER) und ii) Pyrimidin Hydrate, alkylierte Basen und Thymin Glykole iiber den Basen-Exzisions-Reparatur (BER) Mechnismus mit hoher Effizienz reparieren. Die hervorstechendsten Resultate waren, dafi die Reparatur iiber einen distributiven Mechanismus erfolgt und dafi die Poly(ADP-ribosyl)ierung in den zwei untersuchten Reparaturwegen verschieden beteiligt ist. Wahrend der Reparatur von OSO4- und MNNG-Schaden, die iiber den BER Mechanismus erfolgen, wurden Poly(ADPribose) Polymere gebildet, und die Verhinderung der Polymer Synthese fuhrte zu einer drastischen Hemmung der Reparatur. Im Unterschied dazu wurde wahrend der Reparatur von UV- und AAF-Schaden PARP nicht aktiviert und der kompetitive Inhibitor Benzamid hatte keinen EinfluG auf die NER. Diese Ergebnisse implizieren, dafi im Xenopus Ei Lysat System, Poly(ADP-ribosyl)- ierung direkt in der BER aber offensichtlich nicht in der NER beteiligt ist. Dennoch erwies sich selbst die Funktion der PARP in der BER als eine negative, indem das Enzym fest an DNS Strangbriiche bindet und somit Reparaturenzymen den Zugang an die DNS behindert. Erst duch Automodifikation kann PARP von der DNS abgehen und somit Zugang fur Reparaturenzyme schaffen. Xeno pus Lysate mit inaktivierter PARP zeigten voile Reparatur ohne poly(adpribose) Umsatz. Daraus ergibt sich die folgende Frage: "Wenn PARP fur DNS Exzisions-Reparatur nicht benotigt wird, welche Funktion hat es dann bei der zellularen Regeneration nach DNA Schadigung?" Jiingste Daten aus verschiedenen Gruppen, unsere eingeschlossen, fiihrten zu einer neuen Sicht, was die Funktion der Poly(ADP-ribose) betrifft. Es diirfte eine Rolle bei der Verhinde rung von illegitimen Rekombinationsprozessen und im Aufspiiren Schaden spielen. Einig ist man darin, dag Poly(ADP-ribose) von DNS eine Funktion bei der Bewahrung der Genom-Integritat und im Chromatin-Metabolismus spielt. 119
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