Zurich Open Repository and Archive University of Zurich Main Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Year: 2007 Novel enzymes from the radioresistant bacterium Deinococcus radiodurans with potential roles in DNA repair Blasius, Melanie Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-163677 Dissertation Published Version Originally published at: Blasius, Melanie. Novel enzymes from the radioresistant bacterium Deinococcus radiodurans with poten- tial roles in DNA repair. 2007, University of Zurich, Faculty of Science. Novel enzymes from the radioresistant bacterium Deinococcus radiodurans with potential roles in DNA repair Dissertation zur Erlangung der naturwissenschaftlichen Doktorwürde (Dr. sc. nat.) vorgelegt der Mathematisch-naturwissenschaftlichen Fakultät der Universität Zürich von Melanie Blasius aus Deutschland Promotionskomitee Prof. Dr. Ulrich Hübscher (Vorsitz) Prof. Dr. Michael Hottiger Prof. Dr. Josef Jiricny Zürich 2007 M. Blasius: Novel enzymes from the radioresistant bacterium Deinococcus radiodurans 1 1 CONTENTS Page 1 CONTENTS........................……………………………...................................... 1 2 ABSTRACT....................................................…………………………......….... 3 3 ZUSAMMENFASSUNG................................……………………………............ 5 4 ABBREVIATIONS……..............................................………...…………........... .7 5 INTRODUCTION AND REVIEW ARTICLE...............………………….............. 8 5.1. Deinococcus radiodurans- a model organism to study DNA repair….. 8 5.2. Factors that might contribute to radiation resistance……...………....... 9 5.3. DNA repair in bacteria and in D. radiodurans in particular.………….... 11 5.4 Review: Deinococcus radiodurans: How to survive extremes? …........ 14 5.5. DNA repair enzymes characterised in this work……………………….. 30 5.6. Aim of this thesis……………………………………………………........... 33 6 ORIGINAL RESEARCH ARTICLES……………………………………………... 34 6.1 DNA polymerase X from Deinococcus radiodurans possesses a structure-modulated 3’-5’ exonuclease activity involved in radioresistance Molecular Microbiology (2006) 60, 165-176…............. 34 6.2 Enzymes involved in DNA ligation and end-healing in the radioresistant bacterium Deinococcus radiodurans (2007), BMC Molecular Biology, under revision……………………………………………...………………. 47 M. Blasius: Novel enzymes from the radioresistant bacterium Deinococcus radiodurans 2 7 FURTHER UNPUBLISHED DATA………………………………………………. 69 7.1 Selected unpublished results…………………………………………….. 69 7.1.1 Deinococcus radiodurans NAD+-dependent DNA ligation…………………… 69 7.1.2 Deinococcus radiodurans polynucleotide kinase substrate specificity…...... 73 7.1.3 The Deinococcus radiodurans DRB0099 gene product binds ADP-ribose... 75 7.2 Material and Methods………………………………………………….... 76 7.2.1 Antibody production and Western blotting…………………………………….. 76 7.2.2 Preparation of D. radiodurans total extracts………………………………….. 76 7.2.3 DNA ligation assay…………………………………………………………........ 76 7.2.4 DNA binding assay………………………………………………………………. 77 7.2.5 Polynucleotide kinase activity assay…………………………………………... 77 7.2.6 ADP-ribose pull-down assay………………………………………………...…. 78 7.3 Discussion of unpublished results……………………………………….. 78 8 CONCLUSIONS AND PERSPECTIVES..…………………............................... 79 9 REFERENCES (in addition to citations in reprints and manuscripts)………... 83 10 ACKNOWLEDGEMENTS………................…………………………….............. 88 11 CURRICULUM VITAE…..………................…………………………….............. 89 APPENDIX…………………………………………………………………………..……. 91 A Differential incorporation of halogenated deoxyuridines during UV- induced DNA repair synthesis in human cells DNA Repair (2005), 4, 359-366…………………………………….......... 91 M. Blasius: Novel enzymes from the radioresistant bacterium Deinococcus radiodurans 3 2 ABSTRACT Deinococcus radiodurans is a bacterium showing an amazing resistance to ionising radiation. Actively growing cultures show no detectable loss of viability up to 5,000 Gray, which corresponds to approximately 1,600 double-strand breaks per cell. DNA double-strand breaks are the most lethal kind of DNA damage and few double-strand breaks are usually enough to kill a cell. Although the Deinococcus radiodurans genome has been sequenced, it remains unclear which factors contribute to the extremely resistant phenotype. It has been suggested, that the enhanced radioresistance results from an optimal combination of various active and passive mechanisms present in many other radiosensitive organisms. These might include: (i) a potent machinery to deal with oxidative stress, (ii) a robust cell wall, (iii) high levels of Mn(II) to protect proteins and DNA, (iv) mechanisms to prevent degradation of free DNA ends, (v) an ordered structure of the DNA, (vi) genome multiplicity, (vii) a sophisticated regulation of DNA repair, transcription and cell cycle progression and (viii) multiple DNA repair pathways with efficient and specialised enzymes. The aim of this thesis was to identify and characterise novel DNA repair enzymes that are likely to contribute to the extremely high DNA repair capacity of this bacterium. It seems that Deinococcus radiodurans possesses a rather advanced DNA repair apparatus compared to other well-characterised bacteria such as Escherichia coli. Several genes of Deinococcus radiodurans seem to originate from eukaryotes and might have been obtained via horizontal gene transfer. This work focuses on five proteins that are expected to at least partially account for DNA double-strand break repair. In the first part of this work an enzyme has been characterised that is a member of the family X of DNA polymerases. Surprisingly, this protein contains a structure-modulated nuclease activity, suggesting a role in resolving DNA structures that would otherwise prevent further processing by DNA repair enzymes. Deletion of the gene leads to a radiosensitive phenotype with a delay in double-strand break repair. The second part focuses on DNA ligation and end-healing in Deinococcus radiodurans. Characterisation of an NAD+-dependent DNA ligase showed a high ligation activity in the presence of Mn(II) and NAD+. A second ATP-dependent DNA ligase has been predicted by sequence alignments. This enzyme indeed might play a role in DNA repair, as it is part of an operon that is highly upregulated upon irradiation. The recombinant protein possesses adenylyltransferase activity in the presence of ATP and is specifically adenylated on lysine M. Blasius: Novel enzymes from the radioresistant bacterium Deinococcus radiodurans 4 40. However, no DNA ligation activity could be detected under any conditions tested. The second protein of the same putative DNA repair operon turned out to be a polynucleotide kinase that can heal modified DNA termini and prepare them for religation. Mutation of arginine 371 strongly reduces the polynucleotide kinase activity. The third protein of this putative DNA repair operon interestingly shows some sequence similarity to the family of Macro domains. The members of this family are known to bind ADP-ribose monomers and polymers. Indeed a specific binding to ADP-ribose could be detected with this protein. Further work is required to test whether ADP-ribosylation plays a role in bacterial DNA repair and might be involved in signalling, regulation or interactions with other proteins. M. Blasius: Novel enzymes from the radioresistant bacterium Deinococcus radiodurans 5 3 ZUSAMMENFASSUNG Deinococcus radiodurans ist ein Bakterium mit einer sehr hohen Resistenz gegenüber ionisierender Strahlung. Aktiv wachsende Kulturen zeigen keinen messbaren Verlust ihrer Lebensfähigkeit bis zu einer Dosis von 5000 Gray, was ungefähr 1600 Doppelstrangbrüchen in der Desoxyribonukleinsäure (DNS) entspricht. DNS-Doppelstrangbrüche sind die gefährlichsten aller DNA Schäden, und normalerweise sind wenige Doppelstrangbrüche ausreichend, um eine Zelle zu töten. Obwohl das Genom von Deinococcus radiodurans sequenziert ist, weiss man bisher nicht, welche Faktoren zu dieser extremen Radioresistenz beitragen. Es wird angenommen, dass diese auf einer optimalen Kombination verschiedener aktiver und passiver Mechanismen basiert, die auch von anderen nicht radioresistenten Organismen bekannt sind. Dazu gehören vermutlich: (i) eine potente Maschinerie, um den oxidativen Stress zu bewältigen, (ii) eine robuste Zellwand, (iii) ein hoher Gehalt an Mn(II) zum Schutz von Proteinen und DNS, (iv) Mechanismen, die einen Abbau der freien DNS-Enden verhindern, (v) eine geordnete DNS-Struktur, (vi) mehrere Genomkopien, (vii) eine differenzierte Regulation der DNS-Reparatur, der Transkription und des Zellzyklus sowie (viii) diverse Wege zur DNS-Reparatur mit effizienten und spezialisierten Enzymen. Das Ziel dieser Doktorarbeit war die Identifizierung und Charakterisierung neuer DNS- Reparaturenzyme, die wahrscheinlich einen Beitrag leisten können zur extrem hohen DNS- Reparaturkapazität von Deinococcus radiodurans. Anscheinend hat dieses Bakterium einen erweiterten DNS-Reparaturapparat im Vergleich zu anderen gutuntersuchten Bakterien wie beispielsweise Escherichia coli. Mehrere Gene von Deinococcus radiodurans scheinen von Eukaryoten abzustammen und wurden vermutlich über horizontalen Gentransfer übertragen. Fünf Proteine, für die eine Beteiligung an der Doppelstrangbruchreparatur angenommen wird, wurden im Rahmen dieser Dissertation genauer untersucht. Das erste untersuchte Enzym ist ein Mitglied der DNS-Polymerase-Familie X. Überraschenderweise