Biochemical and biophysical characterization of the human TIM/TIPIN/CRY complex - a potential direct link between the circadian clock and the cell cycle Dissertation Zur Erlangung des Grades Doktor der Naturwissenschaften Am Fachbereich Biologie Der Johannes Gutenberg-Universität Mainz Tim Albert Grimmelsmann Mainz, 2020 II III Abbreviations ACN Acetonitrile ADP Adenosine diphosphate AEBSF 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride AEX Anion exchange chromatography APS Ammonium persulfate ATM Ataxia Telangiectasia Mutated ATP Adenosine triphosphate ATR Ataxia telangiectasia and Rad3-related protein BER Base excision repair bHLH Basic-Helix-Loop-Helix BMAL1 Brain and muscle ARNT-like 1 BS3 Bis-sulfosuccinimidyl suberate CAEX Cation exchange chromatography CBD CRY-binding-domain cDNA Complementary DNA CEP131 Centrosomal Protein 131 CHAPS 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate CHK Checkpoint kinase CK Casein kinase CLOCK Circadian Locomotor Output Cycles Kaput CRY Cryptochrome CryoEM Cryogenic electron microscopy CT Circadian time CTD C-terminal domain CV Column volume DDR DNA-damage response DLS Dynamic light scattering Dmax Maximal dimension DMF Dimethyl formamide DMSO Dimethyl sulfoxide DOL Degree of labeling dsDNA Double stranded DNA E-Box Enhancer box EDTA Ethylenediaminetetraacetic acid FA Formic acid FAD Flavin-adenin-dinucleotide FASP Familial advanced sleep phase FBXL3 F-box/Leucine rich repeat protein 3 FDR False discovery rate FP Fluorescence polarization FPC Fork-protection complex FPLC Fast protein liquid chromatography γ-H2AX Phosporylated H2A protein IV GraFix Gradient fixation GRE Glucocorticoid responsive elements HU Hydroxyurea IMAC Immobilized metal ion affinity chromatography IPTG Isopropyl β-D-1-thiogalactopyranoside KD Dissociation constant kDa Kilodalton LB Luria Bertani Medium MALS Multi-angle light scattering MES 2-(N-morpholino)ethanesulfonic acid MOPS 3-(N-morpholino)propanesulfonic acid MS Mass Spectrometry MW Molecular weight MWMALS MW determined by MALS analysis MWPREP MW determined by preparative SEC MWSEC MW determined by analytical SEC NanoSPR Nano Surface plasmon resonance NER Nucleotide excision repair NHEJ Non-homologous end joining NLS Nuclear localization site PARP1 Poly ADP-ribose polymerase 1 PAS PER-ARNT-SIM PBL Phosphate binding loop PBS Phosphate buffered saline PCR Polymerase chain reaction PER Period PHR Photolyase homology region PMSF Phenylmethylsulfonyl fluoride PSM Peptide spectrum matches Rg Radius of gyration Rh Hydrodynamic radius RNAi RNA interference RT Room temperature SAXS Small-angle X-ray scattering SCN Suprachiasmatic nucleus SEC Size exclusion chromatography SILAC Stable Isotope Labeling by Amino acids in Cell culture SLIC Sequence and ligation independent cloning SOC Super optimal broth with added glucose ssDNA Single stranded DNA TAD Transactivation domain TAE Tris-acetate-EDTA buffer TB Terrific broth TCEP Tris(2-carboxyethyl)phosphin TEMED Tetramethylethylenediamine TIM TIMELESS TIPIN TIMELESS-interacting protein V TTFL Transcription-translation feedback loop TTP TIM/TIPIN complex WT Wild-type X-Gal 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside XLMS Crosslink Mass Spectrometry XPA Xeroderma pigmentosum group A YFP Yellow fluorescent protein YLPM1 YLP motif-containing protein 1 VI Table of Contents 1 Zusammenfassung ............................................................................................................. X 2 Abstract ........................................................................................................................... XII 3 Introduction ........................................................................................................................ 1 3.1 Circadian Clocks .......................................................................................................... 1 3.1.1 Molecular clock .................................................................................................... 3 3.1.2 The activating complex ........................................................................................ 5 3.1.3 The repressive complex ........................................................................................ 5 3.1.4 The CRY/PER complex ....................................................................................... 7 3.1.5 Structure of CRY .................................................................................................. 9 3.1.6 Differences between CRY1 and CRY2 .............................................................. 11 3.2 Mammalian TIM ........................................................................................................ 11 3.2.1 DNA Replication ................................................................................................ 12 3.2.2 Replication Stress ............................................................................................... 13 3.2.3 Functions of TIM/TIPIN .................................................................................... 14 3.2.4 TIM/TIPIN in replication stress ......................................................................... 16 3.2.5 Structure of the TIM/TIPIN complex ................................................................. 17 3.3 Connection between the circadian clock, DNA Replication and DNA damage ....... 20 3.4 Aims of the Thesis ..................................................................................................... 22 4 Materials and Methods ..................................................................................................... 23 4.1 Materials .................................................................................................................... 23 4.2 Molecular Biology Methods ...................................................................................... 29 4.2.1 Preparation of competent cells ........................................................................... 30 4.2.2 Transformation ................................................................................................... 30 4.2.3 Amplification and Isolation of plasmids ............................................................ 31 4.2.4 Sequence and ligation independent cloning ....................................................... 31 4.2.5 PCR .................................................................................................................... 31 4.2.6 Recombination of Insert and Vector .................................................................. 33 4.2.7 Colony PCR ........................................................................................................ 33 4.2.8 Agarose Gel Electrophoresis .............................................................................. 34 4.2.9 Protein expression in E. coli ............................................................................... 34 4.2.10 Protein expression in Insect cells ....................................................................... 35 4.3 Biochemical Methods ................................................................................................ 39 4.3.1 Determination of Protein and DNA concentrations ........................................... 39 4.3.2 Gel electrophoresis ............................................................................................. 40 VII 4.3.3 Protein purification ............................................................................................. 41 4.3.4 Fluorescence polarization ................................................................................... 46 4.3.5 Nanoparticle Surface Plasmon Resonance (NanoSPR) ..................................... 48 4.3.6 Analytical SEC ................................................................................................... 50 4.3.7 SAXS (small angle x-ray scattering), MALS (multi angle light scattering) and DLS (dynamic light scattering) ........................................................................................ 50 4.3.8 Protein pulldowns of CRY with TIM-N-∆L/SL ................................................ 52 4.3.9 Pulldowns with TTP/TTP-∆L using SILAC extract and MS ............................. 53 4.3.10 Crosslink Mass Spectrometry ............................................................................. 54 4.3.11 Crosslinking of TTP/CRY2 by Gradient Fixation for single particle EM ......... 56 5 Results .............................................................................................................................. 58 5.1 Protein Purifications .................................................................................................. 58 5.1.1 CRY1 .................................................................................................................. 59 5.1.2 CRY2 .................................................................................................................. 59 5.1.3 Purification of C-terminal CRY tails ................................................................. 60 5.1.4 Purification of TIMELESS/TIPIN ..................................................................... 62 5.1.5 TIM-N-∆L .......................................................................................................... 63 5.1.6 TIM-N-Sumo ...................................................................................................... 64 5.1.7 TIPIN .................................................................................................................