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Investigation of Structure and Function of Esco1 and Esco2 Acetyltransferases

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Investigation of Structure and Function of Esco1 and Esco2 Acetyltransferases Investigation of Structure and Function of Esco1 and Esco2 Acetyltransferases Dissertation for the award of the degree “Doctor rerum naturalium” of the Georg-August-Universität Göttingen within the doctoral program “Biology” of the Georg-August University School of Science (GAUSS) submitted by Tahereh Ajam from Iran Göttingen, 2018 Thesis Committee Prof. Dr. Gregor Eichele, Genes and Behavior, Max-Planck-Institute for Biophysical Chemistry, Göttingen (Mentor and Reviewer) Dr. Martin Kollmar, Systems Biology of Motor Proteins, Max-Planck-Institute for Biophysical Chemistry, Göttingen (Co-reviewer) Members of the Extended Examination Board Dr. Vladimir Pena, Macromolecular Crystallography, Max-Planck-Institute for Bio- physical Chemistry, Göttingen Prof. Dr. Ahmed Mansouri, Molecular Cell Differentiation, Max-Planck-Institute for Biophysical Chemistry, Göttingen Prof. Dr. Henning Urlaub, Bioanalytical Mass Spectrometry, Max-Planck-Institute for Biophysical Chemistry, Göttingen Prof. Dr. Steven Johnsen, Translational Cancer Research, University Medical Center Göttingen, Göttingen Date of oral examination: 22.11.2018 Affidavit I hereby declare that the dissertation entitled “Investigation of Structure and Func- tion of Esco1 and Esco2 Acetyltransferases” is submitted in support of my applica- tion for the degree of Doctor rerum naturalium to Georg-August-Universität Göttin- gen within the doctoral program Biology of the Georg-August University School of Science (GAUSS). This thesis has been written independently and with no sources and aids other than quoted within texts, references and acknowledgments. This thesis has not been submitted in any previous application for any degree, elsewhere. Göttingen, October 2018 Tahereh Ajam 3 To my mom for her boundless love 4 Table of Contents Affidavit .............................................................................................................................................3 Summary ............................................................................................................................................9 Introduction .............................................................................................................................. 11 Functions and regulation of cohesin ................................................................................... 11 1.1.1 Cohesin and the cell cycle .......................................................................................... 11 1.1.2 Cohesin complex architecture .................................................................................... 12 1.1.3 Cohesin functions ...................................................................................................... 14 1.1.4 Cohesin loading and unloading .................................................................................. 15 1.1.5 Cohesion establishment .............................................................................................. 17 1.1.6 The prophase pathway of cohesin dissociation............................................................ 19 Smc3 acetylation by Esco1 and Esco2 ............................................................................... 21 1.2.1 Esco1 and Esco2 belong to GCN5 family ................................................................... 21 1.2.2 GNAT family structure, acetyl-CoA binding and substrate recognition ....................... 22 1.2.3 GNAT family catalysis mechanism ............................................................................ 23 Aims of the study .............................................................................................................. 26 Material and Methods................................................................................................................ 28 Antibodies ......................................................................................................................... 28 Protein expression in insect cells ........................................................................................ 28 2.2.1 Cloning and mutagenesis ........................................................................................... 28 2.2.2 Baculovirus generation............................................................................................... 30 5 2.2.3 Protein expression in insect cells ................................................................................ 31 Protein purification ............................................................................................................ 32 2.3.1 Purification of mouse Esco2368-592............................................................................... 32 2.3.2 Purification of full-length human ESCO1 ................................................................... 33 2.3.3 Purification of trimeric and tetrameric cohesin complex ............................................. 34 Characterization of purified proteins .................................................................................. 35 2.4.1 SDS-polyacrylamide gel electrophoresis (SDS-PAGE)............................................... 35 2.4.2 Protein staining .......................................................................................................... 35 2.4.3 Protein concentration measurement ............................................................................ 35 Crystallization and structure determination ........................................................................ 36 In vitro acetylation assay ................................................................................................... 36 Cell culture, transfection and synchronization .................................................................... 37 Flow cytometry ................................................................................................................. 38 Cell extract preparation and immunoblotting ...................................................................... 38 Prometaphase chromosome spreads, cytospin and immunofluorescence analysis ................ 39 Statistical analysis ............................................................................................................. 40 Results ...................................................................................................................................... 41 Expression, purification and crystallization of the MmEsco2 protein .................................. 41 Overall structure of the MmEsco2368-592 in complex with Coenzyme A (CoA) .................... 46 Active site architecture and implications for catalysis ........................................................ 48 6 Effect of site-directed mutagenesis of the active site residues on acetyltransferase activity of Esco1 and Esco2 ........................................................................................................................... 52 3.4.1 Expression and purification of HsESCO1 and HsESCO2 ........................................... 52 3.4.2 Expression and purification of trimeric and tetrameric cohesin complexes .................. 54 3.4.3 Establishment of in vitro cohesin acetylation by HsESCO1 ........................................ 56 3.4.4 In vitro analysis of active site mutants of HsESCO1 ................................................... 58 Effect of catalytic site mutants of Esco1 and Esco2 on in vivo acetyltransferase activity ..... 59 3.5.1 Effect of catalytic site mutants of MmEsco1 on Smc3 acetylation in vivo ................... 60 3.5.2 Effect of catalytic site mutants of MmEsco2 on Smc3 acetylation .............................. 63 Effect of catalytic site mutants of MmEsco2 on sister chromatid cohesion ......................... 66 Discussion................................................................................................................................. 69 The architecture of MmEsco2 in complex with CoA .......................................................... 69 Characterization of the active site of Esco1 and Esco2 ....................................................... 71 4.2.1 In vitro cohesin acetylation requires ATP hydrolysis and DNA .................................. 71 4.2.2 In vitro analysis of active site mutants of HsESCO1 ................................................... 71 4.2.3 In vivo analysis of active site mutants of MmEsco1 .................................................... 72 4.2.1 In vivo analysis of active site mutants of MmEsco2 .................................................... 73 Structural interpretation of Roberts syndrome mutations .................................................... 75 Additional Data ......................................................................................................................... 77 MmEsco2368-592 shows inefficient autoacetyltransferase activity.......................................... 77 HsESCO2 does not acetylate the trimer under in vitro conditions ....................................... 78 7 In vitro cohesin acetylation shows salt sensitivity ............................................................... 79 S809 and D810 are crucial for the autoacetylation activity of HsESCO1 under in vitro conditions ..................................................................................................................................... 80 References .......................................................................................................................................
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