FACULTY OF SCIENCE UNIVERSITY OF COPENHAGEN DENMARK PhD Thesis Katrine Vyff Løvschal Regulation of Ribonucleotide Reductase and Screening for Species-Specific Inhibitors in Fission Yeast Academic advisor: Associate Professor Christian Holmberg Submitted: 21/03/2019 Regulation of Ribonucleotide Reductase and Screening for Species-Specific Inhibitors in Fission Yeast Katrine Vyff Løvschal PhD Thesis March 2019 This thesis has been submitted to the PhD School of Science at the University of Copenhagen, Denmark Preface This thesis has been submitted to the PhD School of Science at the University of Copenhagen. The research presented herein was carried out between March 2016 and March 2019 in the Cell Cycle and Genome Stability Lab, Department of Biology, University of Copenhagen under supervision of Associate Professor Christian Holmberg. The thesis covers two main areas presented in two chapters. Chapter 1 provides a general introduction to the cellular functions of eukaryotic class Ia ribonucleotide reductases. It explores the genetic interactions involving ribonucleotide reductase and genome stability factors in fission yeast, most of which are conserved in humans. Chapter 2 presents a strategy to screen for novel and species-specific inhibitors of ribonucleotide reductase using fission yeast. This thesis presents original, unpublished work, except where references are made to previous work. In addition to my work presented in the two chapters, I have contributed to the following publication: Paper I Deoxynucleoside Salvage in Fission Yeast Allows Rescue of Ribonucleotide Reductase Deficiency but Not Spd1-Mediated Inhibition of Replication Oliver Fleck, Ulrik Fahnøe, Katrine Vyff Løvschal, Marie-Fabrice Uwamahoro Gasasira, Irina N. Marinova, Birthe B. Kragelund, Antony M. Carr, Edgar Hartsuiker, Christian Holmberg and Olaf Nielsen Genes (2017) 8(5), DOI: 10.3390/genes8050128 v Acknowledgements First of all, I would like to thank Associate Professor Christian Holmberg and Professor Olaf Nielsen for the provision of the laboratory facilities in the Cell Cycle and Genome Stability Lab and for creating an inspiring research environment. I also wish to thank the Villum Foundation for funding my PhD project. I am sincerely grateful to my supervisor Christian Holmberg for sharing his expertise and providing me with continued guidance throughout my studies, as well as for commenting on this thesis. I also wish to express my gratitude for the opportunity to attend the 9th International Fission Yeast Meeting in Banff, Alberta, Canada and the Yeast Genetics and Genomics course in Cold Spring Harbor, New York, USA. Olaf Nielsen is thanked for assisting as co-supervisor and always being helpful and open for discussions. I would also like to thank past and present members of the Cell Cycle and Genome Stability Lab, as well as former and current office mates for creating a wonderful work environment and for inspiring discussions. I would especially like to thank Michaela Rasmussen for her technical assistance in the lab. Finally, I take this opportunity to express my gratitude to my wonderful family and friends for always being there for me with kind words and inspirational patience. Thank you, Andreas, for your unconditional love and support. I could not have done this without you. vii Contents Preface …………………………………………………………………………………………….…...v Acknowledgements………………………………………………………………………………..vii Abstract………………………………………………………………………………………...……..ix Resumé………………………………………………………………………………………...……..xi Abbreviations…………………………………………………………………………………….....xv Chapter 1 Aspects of Ribonucleotide Reductase and Species-specific Regulation……………...1 Introduction……………………………………………………………………………….…...3 Schizosaccharomyces pombe……………………………………………………….3 Life cycle……………………………………………………………..………....4 Mating types………………………………………...…………………….……5 S. pombe as model organism…………………………………………….….7 DNA integrity checkpoints and genome stability………………………….…..8 Ribonucleotide reductase……………………………………………………….….11 Regulation of ribonucleotide reductase…………………………………………16 Transcriptional and post-translational regulation...……………….…..16 Regulation by allostery and oligomerization...………………………...17 Regulation by small protein inhibitors…………………………………..21 Ubiquitin-mediated regulation of RNR inhibitors…………………….23 Cdt2 CRL4 inactivation……………………………………………………….25 Results………………………………………………….………………..……………………27 Generation of fission yeast strains relying on human RNR……….…….…27 Strain characterization.……………..……….………………..……………………29 The effect of spd1 overexpression on cell cycle progressio………………..33 Human RNR in fission yeast confers checkpoint activation and dependency…………………………………….………………..……………………35 Elevated levels of the R2 homologs suppress checkpoint activation and dependency…………………………………….………………..……………………38 In vivo function(s) of Spd1…………………………………….………...………..42 Spd1 is destabilized in cells with human RNR……………………………….47 Discussion……………………………………………………………………………….......50 Chapter 2 Co-culturing System to Screen for Species-Specific Inhibitors of RNR………….59 Introduction……………………………………………………………………………….....61 Results………………………………………………………………………………...……...63 Experimental setup…………………………………………………………….…...63 Strain creation…………………………………………………………………….....65 Proof of principle…………………………………………………………….……..66 Discussion and future perspectives…………………………………………………….69 Materials and Methods………………………………………………………………………...…73 References………………………………………………………………………………………......89 Paper 1………………………………………………………………..……………………………...97 Appendix – Supplementary Figures………………………………………………………….113 Abbreviations aa − amino acid dGTP − deoxyguanosine triphosphate Amp − ampicillin Dif1 − damage-regulated import facilitator 1 APC − anaphase promoting complex DmdNK − Drosophila melanogaster ATM − ataxia-telangiectasia mutated deoxyribonucleoside kinase ATP − adenosine triphosphate DMSO − dimethyl sulfoxide ATR − ataxia telangiectasia and Rad3- DNA − deoxyribonucleic acid related dNDP − deoxynucleoside diphosphate BF − bright field dNTP − deoxyribonucleoside triphosphate bp − base pairs dTTP − deoxythymidine phosphate BSA − bovine serum albumin DSB − double-strand break C. albicans − Candida albicans Dun1 − DNA-damage uninducible 1 Cdc − cell division cycle E. coli − Escherichia coli cDNA − complementary DNA EtOH − ethanol Cdh1 − cadherin 1 FACS − fluorescence-activated cell sorting Cds1 − checking DNA synthesis 1 G418 − genetecin Cdt2 − Cdc-10-dependent transcript 2 G1 − gap 1 CFP − cyan fluorescent protein G2 − gap 2 ChimR2 − chimeric ribonucleotide reductase h − hour small subunit h+/- − heterothallic Chk − checkpoint kinase h90 − homothallic COP9 − constitutive photomorphogenesis 9 HA − hemagglutinin CPT − camptothecin hENT1 − human equilibrative nucleoside CRL − cullin RING E3 ligase transporter 1 CSN − COP9 signalosome hRNR − human ribonucleotide reductase C-terminal − carboxy terminal HRP − horse radish peroxidase Cul4 − cullin 4 HU − hydroxyurea cut − cell untimely torn IDP − intrinsically disordered protein dATP − deoxyadenosine triphosphate IgG − immunoglobulin G dCTP − deoxycytidine triphosphate IP − immunoprecipitation Ddb1 − DNA-damage binding 1 xi IRBIT − IP3R binding protein released with Pol − polymerase inositol 1,4,5-triphosphate R1 − ribonucleotide reductase large subunit Kana − kanamycin R2 − ribonucleotide reductase small subunit KanMX − kanamycin A selection marker Rad − radiation sensitive kDa − kilodalton RLU − relative light units LiOAc − lithium acetate RNA − ribonucleic acid MBF − Mlu1 binding factor RNR − ribonucleotide reductase MBP − myelin basic protein ROS − reactive oxygen species mCherry − monomeric derivative of DsRed RPA − replication protein A fluorescent protein RRM1 − ribonucleotide reductase catalytic Mec1 − mitosis entry checkpoint 1 subunit M1 [Homo sapiens] Mik1 − mitosis inhibitory kinase 1 RRM2 − ribonucleotide reductase regulatory MMS − methyl methanesulfonate subunit M2 [Homo sapiens] MSA − minimal sporulating agar RRM2B − ribonucleotide reductase MSL − minimal sporulation liquid regulatory TP53 inducible subunit M2B Mbu1 − multi budding 1 [Homo sapiens] NDP − ribonucleoside diphosphate RT − room temperature NDPK − nucleoside diphosphate kinase S. cerevisiae − Saccharomyces cerevisiae NER − nucleotide excision repair S. pombe − Schizosaccharomyces pombe NLS − nuclear localization sequence SCF − Skp1-Cul1-F-box nmt − no message in thiamine S.d. − standard deviation N-terminal − amino terminal SDS-PAGE − Sodium dodecyl sulphate O/N − overnight polyacrylamide gel electrophoresis ORF − open reading frame siRNA − small interfering RNA P. falciparum − Plasmodium falciparum Sml1 − suppressor of Mec1 lethality 1 PCNA − proliferating cell nuclear antigen Spd1 − S phase delayed 1 PCR − polymerase chain reaction ssDNA − single-stranded DNA p53 − tumor protein 53 (TP53) Ste9 − sterile 9 p53R2 − p53-inducible ribonucleotide Suc22 − suppressor of cdc22ts reductase small subunit homolog TCA − trichloracetic acid PIKK − phosphatidylinositol 3-kinase- TMPK − thymidylate phosphate kinase related kinase ts − temperature sensitive xii TS − thymidylate synthase Wt − wild type Ub − ubiquitin YES − yeast extract solid Ubr2 − E3 ubiquitin-protein ligase YEL − yeast extract liquid UV − ultraviolet xiii Abstract Cellular proliferation and genome integrity depend on accurate DNA replication and repair of damaged DNA, which in turn are impacted by the level of deoxyribonucleotides. Ribonucleotide reductase is the enzyme responsible for the de novo synthesis of deoxyribonucleotides in all organisms and is thus