Corso di dottorato di ricerca in Scienze biomediche e biotecnologiche Ciclo XXXI Titolo della tesi “Role of Ape1 acetylation in fine tuning its activity on telomeric regions” Dottoranda Supervisore Dott.ssa Silvia Burra Prof. Gianluca Tell Anno di discussione 2018-2019 Niente nella vita va temuto, dev’essere solamente compreso. Ora è tempo di comprendere di più, così possiamo temere di meno. Marie Curie 2 Summary Summary ........................................................................................................................... 3 1. Abstract ...................................................................................................................... 5 2. Abbreviations ............................................................................................................ 6 3. Figures index ............................................................................................................. 7 4. Introduction ............................................................................................................... 9 4.1. Cancer cells features .......................................................................................... 9 4.2. Base Excision Repair (BER) pathway ............................................................. 11 4.3. Apurinic/apyrimidinic endonuclease 1 (Ape1) ................................................ 12 4.4. Telomeres organization .................................................................................... 21 4.5. Ape1 role at the telomeres ................................................................................ 33 5. Aim of this study ..................................................................................................... 36 6. Results ..................................................................................................................... 37 6.1. Structural analysis of G4 sequences by circular dichroism (CD) spectroscopy 37 6.2. The 33-residue Ape1 N-terminus is required to stably bind telomeric G4 structures ..................................................................................................................... 40 6.3. The enzymatic activity of Ape1 on G4 telomeric structures is influenced by ionic strength and the N-terminus ............................................................................... 44 6.4. Acetylatable lysine residues in the Ape1 N-terminus are required for binding and enzymatic activity on G4 telomeric structures ..................................................... 53 6.5. Activity of Ape1 protein variants from U2OS cells ......................................... 56 6.6. The telomeric-binding ability of Ape1 depends on K27, K31, K32 and K35 residues ........................................................................................................................ 61 6.7. Interaction of NPM1 with Ape1 modulates its endonuclease activity on telomeric sequences .................................................................................................... 62 ........................................................................................................................................ 66 7. Discussion ................................................................................................................ 67 8. Future perspectives .................................................................................................. 72 9. Materials and Methods ............................................................................................ 74 9.1. Protein expression and FPLC purification ....................................................... 74 9.2. Oligonucleotides synthesis, purification and annealing ................................... 74 9.3. Circular dichroism (CD) spectroscopy ............................................................. 75 9.4. SDS-PAGE and Western blotting .................................................................... 76 9.5. Electrophoretic mobility shift assay (EMSA) .................................................. 77 9.6. UV-crosslinking experiments ........................................................................... 77 3 9.7. Cell cultures, transfection and Co-immunoprecipitation ................................. 77 9.8. Chromatin immunoprecipitation and quantitative PCR ................................... 78 9.9. AP site incision assays ..................................................................................... 79 9.10. Surface Plasmon Resonance (SPR) experiments ......................................... 80 9.11. Telomeric length assay ................................................................................. 80 9.12. GST pull-down assay ................................................................................... 80 9.13. Statistical analysis ........................................................................................ 81 10. References ............................................................................................................ 82 11. Published articles ............................................................................................... 101 12. Acknowledgements ............................................................................................ 102 4 1. Abstract One of the hallmarks of cancer is the loss of telomere stability. Unprotected telomeres are prone to aberrant end-joining reactions that lead to chromosomal fusions and translocations. Human telomeres are formed by the repeated TTAGGG sequence, in which the 3’ exposed strand may adopt a G-quadruplex (G4) structure. The guanine-rich regions of telomeres are hotspots for oxidation forming 8-oxoguanine, a lesion that is handled by the base excision repair (BER) pathway. One key player of this pathway is Ape1, the main human endonuclease processing abasic sites. Recent evidences showed an important role for Ape1 in telomeric physiology, but the molecular details regulating Ape1 enzymatic activities on G4 telomeric sequences are lacking. Through a combination of in vitro assays, we demonstrate that Ape1 can bind and process different G4 structures containing an abasic site analog (THF) in two different locations and that the interaction involves specific acetylatable lysine residues (i.e. K27/K31/K32/K35) present in the unstructured N-terminal domain of the protein. The cleavage of the abasic site located in a G4 structure by Ape1 depends on the DNA conformation or the position of the lesion and on electrostatic interactions between the protein and the nucleic acids. Moreover, Ape1 mutants mimicking the acetylated protein display increased cleavage activity for abasic sites. We found that nucleophosmin (NPM1), which binds the N-terminal domain of Ape1, plays a role in modulating telomere length and Ape1 activity at abasic G4 structures. Thus, the Ape1 N-domain is an important relay site for regulating the enzyme’s activity on G4-telomeric sequences, and specific acetylatable lysine residues constitute key regulatory sites of Ape1 enzymatic activity dynamics at telomeres. 5 2. Abbreviations ALT Alternative Lengthening of Telomeres AP Apurinic/apyrimidinic site Ape1 AP endonuclease 1 BER Base Excision Repair F Tetrahydrofuran G4 G-quadruplex OG 8-oxo-7,8-dihydroGuanine Lys Lysine nCaRE negative Calcium Responsive Element NPM1 Nucleophosmin 1 PTM Post-Translational Modification ROS Reactive Oxygen Species TERC Telomere RNA Component TERT Telomere Reverse Transcriptase TERRA Telomeric Repeat-containing RNA TRF Telomeric Repeat binding Factor 6 3. Figures index Figure 1 The hallmarks of cancer ............................................................................................... 9 Figure 2 Overview of different damaging agents, DNA effects and relative DNA repair pathways .................................................................................................................................... 10 Figure 3 Graphical representation of the main BER steps .................................................... 11 Figure 4 Structural organization of Ape1 protein .................................................................. 13 Figure 5 Cartoon representation of the Ape1-DNA complex ................................................. 14 Figure 6 Representation of the product of guanine oxidation ............................................... 17 Figure 7 Ape1 protein network ................................................................................................. 20 Figure 8 Representation of the chromosome ends protected by members of the shelterin complex ...................................................................................................................................... 22 Figure 9 Representation of a planar tetrad ............................................................................. 25 Figure 10 Possible G-quadruplex structure conformations ................................................... 25 Figure 11 Mechanisms of telomerase-driven (A) and HR-driven (B) synthesis of telomeric DNA ............................................................................................................................................ 27 Figure 12 Protein interactome network of telomeric partners .............................................. 34 Figure 13 CD spectroscopy of the oligonucleotides used in this study .................................. 38 Figure 14 Schematic representations of G-quadruplex structures assumed by the oligonucleotides