INAUGURAL - DISSERTATION zur Erlangung der Doktorwürde der Naturwissenschaftlich-Mathematischen Gesamtfakultät der Ruprecht - Karls - Universität Heidelberg Vorgelegt von Dipl.-Biol. Mark Bohlmann aus Hamburg Tag der mündlichen Prüfung: Characterization of the SMC-hinge and MutL-homology domain (SMUDO) protein Gutachter: Prof. Dr. Rainer Zawatzky Dr. Thomas G. Hofmann Meinen Eltern Contents 1 Abstract ............................................................................................................................................................. 1 2 Zusammenfassung .................................................................................................................................. 2 3 Introduction .................................................................................................................................................. 4 3.1 Cancer & DNA damage ........................................................................................................................... 4 3.2 The DNA damage response ................................................................................................................. 5 3.3 HIPK2 .............................................................................................................................................................. 7 3.4 The SMC protein family ......................................................................................................................... 9 3.5 MutL proteins and their functions ................................................................................................13 3.6 SMUDO homologs ...................................................................................................................................15 3.7 Task of this thesis ...................................................................................................................................17 4 Materials and Methods .....................................................................................................................18 4.1 General .........................................................................................................................................................18 4.2 Methods in microbiology ....................................................................................................................18 4.2.1Transformation of chemical competent cells ......................................................................... 18 4.2.2 Preparation of bacterial cultures ................................................................................................ 18 4.2.3 Screening of recombinants for fusion protein expression ............................................... 19 4.2.4 Production of recombinant fusion proteins ........................................................................... 19 4.3 Methods in cell biology ........................................................................................................................19 4.3.1 Cell culture ........................................................................................................................................... 19 4.3.2 Cryoconservation .............................................................................................................................. 20 4.3.3 Transient transfection of eukaryotic cells .............................................................................. 20 4.3.4 Lentiviral transduction of eukaryotic cell ............................................................................... 22 4.3.5 Treatment of eukaryotic cells ...................................................................................................... 22 4.3.6 Colony Formation Assay ................................................................................................................ 23 4.4 Methods in molecular biology .........................................................................................................23 4.4.1 Plasmid preparation ........................................................................................................................ 23 4.4.2 Agarose gel electrophoresis ......................................................................................................... 23 4.4.3 DNA digestion and molecular cloning ...................................................................................... 24 I 4.4.4 RNA isolation ...................................................................................................................................... 24 4.4.5 Reverse transcription ...................................................................................................................... 24 4.4.6 DNA amplification by polymerase chain reaction (PCR) .................................................. 25 4.4.7 Generation of expression constructs ........................................................................................ 25 4.4.8 Northern blot analysis .................................................................................................................... 26 4.4.9 Yeast two-hybrid screening .......................................................................................................... 26 4.4.10 DNA sequencing .............................................................................................................................. 26 4.5 Microscopy .................................................................................................................................................26 4.5.1 Immunofluorescence microscopy .............................................................................................. 26 4.5.2 Fluorescence recovery after photobleaching (FRAP) ........................................................ 27 4.6 Bioinformatics..........................................................................................................................................28 4.7 Methods in protein biochemistry ..................................................................................................28 4.7.1 Preparation of total cell lysates ................................................................................................... 28 4.7.2 Determination of protein concentration and purity ........................................................... 29 4.7.3 Subcellular fractionation ............................................................................................................... 29 4.7.4 SDS-polyacrylamide gel electrophoresis ................................................................................. 29 4.7.5 Coomassie blue staining ................................................................................................................. 30 4.7.6 Drying of SDS-polyacrylamide gels ............................................................................................ 30 4.7.7 Western blot (Immunoblotting) ................................................................................................. 30 4.7.8 Purification of recombinant proteins ....................................................................................... 31 4.7.9 Immunoprecipitation and Co-Immunoprecipitation ......................................................... 32 4.7.10 In vitro Translation of 35S-labeled Proteins ......................................................................... 32 4.7.11 GST pulldown assay ...................................................................................................................... 32 4.7.12 In vitro phosphorylation experiments ................................................................................... 33 4.8 Materials .....................................................................................................................................................34 4.8.1 Materials and Kits ............................................................................................................................. 34 4.8.2 Chemicals ............................................................................................................................................. 35 4.8.3 Proteins and enzymes ..................................................................................................................... 36 4.8.4 Markers ................................................................................................................................................. 37 4.8.5 Buffers, Solutions and Media ........................................................................................................ 37 4.8.6 Eukaryotic cell lines ......................................................................................................................... 43 4.8.7 Bacterial strains ................................................................................................................................. 44 4.8.9 cDNA clones ........................................................................................................................................ 44 4.8.10 Expression vectors ........................................................................................................................ 44 4.8.11 Synthetic oligonucleotides ......................................................................................................... 45 4.8.12 SMUDO Northern blot probe ....................................................................................................