Biochemical and functional characterization of the Pch2/ORC AAA+ assembly in controlling meiotic DNA break formation Inaugural-Dissertation zur Erlangung des Doktorgrades Dr. rer. nat. der Fakultät für Biologie an der Universität Duisburg-Essen vorgelegt von María Ascensión Villar Fernández aus Sevilla, Spanien durchgeführt am Max-Planck-Institut für molekulare Physiologie Abteilung für mechanistische Zellbiologie Dezember 2019 Index I Die der vorliegenden Arbeit zugrunde liegenden Experimente wurden am Max-Planck-Institut für molekulare Physiologie in der Abteilung für mechanistische Zellbiologie durchgeführt. 1. Gutachter: Prof. Dr. Andrea Musacchio 2. Gutachter: Prof. Dr. Dominik Boos Vorsitzender des Prüfungsausschusses: Prof. Dr. Stefan Westermann Tag der mündlichen Prüfung: 25. Februar 2020 Diese Dissertation wird über DuEPublico, dem Dokumenten- und Publikationsserver der Universität Duisburg-Essen, zur Verfügung gestellt und liegt auch als Print-Version vor. DOI: 10.17185/duepublico/72234 URN: urn:nbn:de:hbz:464-20200722-140752-1 Alle Rechte vorbehalten. Index II Im Zuge dieser Doktorarbeit wurde der folgende Artikel veröffentlicht: (In the context of this doctoral work, the following article was published:) María Ascensión Villar-Fernández, Richard Cardoso da Silva, Dongqing Pan, Elisabeth Weir, Annika Sarembe, Vivek B. Raina, John R. Weir, Gerben Vader. (2019). A meiosis-specific AAA+ assembly reveals repurposing of ORC during budding yeast gametogenesis. BioRxiv, doi: https://doi.org/10.1101/598128 Index III Content List of Figures ...................................................................................................................... VIII List of Tables ............................................................................................................................ X List of Abbreviations .............................................................................................................. XI 1 Introduction ................................................................................................... 1 1.1 An overview of meiosis ............................................................................................ 1 1.1.1 Meiotic cell cycle phases ............................................................................. 7 1.1.1.1 G1 phase ........................................................................................ 7 1.1.1.2 S phase ........................................................................................... 9 1.1.1.3 G2/ Prophase I ............................................................................. 11 1.1.1.4 Meiosis I ...................................................................................... 14 1.1.1.5 Meiosis II ..................................................................................... 16 1.1.2 Checkpoints ................................................................................................ 17 1.2 Meiotic recombination .......................................................................................... 20 1.2.1 DSB formation and inter-homolog repair ............................................... 20 1.2.2 Non-random spatial distribution of DSBs across the genome ............... 25 1.3 Repetitive elements of the DNA and heterochromatin: the ribosomal DNA (rDNA) as a model of repetitive DNA ................................................................ 29 1.3.1 Repetitive DNA and principles of heterochromatin formation ........... 29 1.3.2 Ribosomal DNA (rDNA) ........................................................................... 33 1.3.2.1 Structure, function and silencing of the rDNA ............................ 33 1.3.2.2 The integrity of the rDNA and rDNA amplification system ....... 36 1.4 AAA+ ATPases ....................................................................................................... 38 1.4.1 Pch2 as a master regulator in meiosis and mitosis ................................. 43 1.4.1.1 Pch2 in meiotic G2/prophase ....................................................... 43 1.4.1.2 Pch2TRIP13 in the mitotic checkpoint ............................................ 52 1.4.2 ORC ............................................................................................................ 54 1.4.2.1 Structural organization of the ORC subunits ............................... 55 1.4.2.2 ORC in DNA replication ............................................................. 58 1.4.2.2.1 Main principles of ORC as an initiator of DNA replication .................................................................... 58 1.4.2.2.2 Origins of replication and insights on ORC structure . 59 1.4.2.2.3 Mechanism of the eukaryotic DNA replication: MCM loading and MCM activation ...................................... 62 1.4.2.2.4 Control of DNA replication ........................................ 65 1.4.2.2.5 Role of the chromatin structure in ORC association with origins of replication ........................................... 67 1.4.2.2.6 Pre-meiotic replication ................................................ 70 1.4.2.3 Other functions of ORC ............................................................... 71 1.5 Objectives ............................................................................................................... 73 Index IV 2 Material and Methods ................................................................................ 75 2.1 Materials ................................................................................................................ 75 2.1.1 Chemicals and reagents ............................................................................ 75 2.1.2 Enzymes and commercial mixes .............................................................. 78 2.1.3 Commercial kits ......................................................................................... 78 2.1.4 Antibiotics .................................................................................................. 79 2.1.5 Antibodies .................................................................................................. 79 2.1.6 Buffers and solutions ................................................................................. 80 2.1.7 Media .......................................................................................................... 83 2.1.8 Synthetic oligonuocleotides ....................................................................... 84 2.1.9 Plasmids ...................................................................................................... 85 2.1.10 Competent cells .......................................................................................... 85 2.1.11 Yeast strains ............................................................................................... 85 2.1.12 Laboratory instruments and devices ....................................................... 85 2.1.13 Online tools ................................................................................................ 89 2.1.14 Software ...................................................................................................... 89 2.2 Methods .................................................................................................................. 90 2.2.1 Growth and maintenance of Saccharomyces cerevisiae ......................... 90 2.2.1.1 Growth conditions of yeast strains .............................................. 90 2.2.1.2 Meiotic cell cycle synchronization .............................................. 90 2.2.1.3 Yeast stock maintenance .............................................................. 91 2.2.2 Construction of yeast strains .................................................................... 91 2.2.2.1 Yeast transformation .................................................................... 91 2.2.2.2 Creation of diploid yeast strains .................................................. 93 2.2.2.3 Sporulation of diploid cells .......................................................... 93 2.2.2.4 Preparation and dissection of tetrads ........................................... 94 2.2.2.5 Determination of phenotype by replica plating ........................... 94 2.2.2.6 Determination of mating type ...................................................... 95 2.2.3 Yeast two-hybrid system ........................................................................... 95 2.2.4 Cloning and methods of DNA analysis (Molecular biology methods) .. 96 2.2.4.1 Polymerase Chain Reaction (PCR) .............................................. 96 2.2.4.2 Cloning using restriction enzymes .............................................. 98 2.2.4.2.1 Agarose gel electrophoresis ........................................ 98 2.2.4.2.2 Restriction digestion ................................................... 99 2.2.4.2.3 DNA extraction and purification ................................. 99 2.2.4.2.4 Ligation ..................................................................... 100 2.2.4.3 Restriction free cloning (Gibson assembly) .............................. 100 2.2.4.4 biGBac method .......................................................................... 101 2.2.4.5 Site-directed mutagenesis .......................................................... 103 2.2.4.6 Transformation of chemically competent bacterial cells ........... 103 2.2.4.7 Isolation of plasmids from bacterial cells .................................. 104 2.2.4.8 Determination of DNA