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Biochemical Characterization of FAN1 Nuclease

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Biochemical Characterization of FAN1 Nuclease Zurich Open Repository and Archive University of Zurich Main Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Year: 2016 Biochemical Characterization of FAN1 Nuclease Pizzolato, Julia Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-128687 Dissertation Originally published at: Pizzolato, Julia. Biochemical Characterization of FAN1 Nuclease. 2016, University of Zurich, Faculty of Science. Biochemical Characterization of FAN1 Nuclease Dissertation zur Erlangung der naturwissenschaftlichen Doktorwürde (Dr. sc. nat.) vorgelegt der Mathematisch-naturwissenschaftlichen Fakultät der Universität Zürich von Julia Pizzolato von Zürich, ZH Promotionskomitee Prof. Dr. Josef Jiricny (Vorsitz, Leitung der Dissertation) Prof. Dr. Alessandro A. Sartori Prof. Dr. Orlando D. Schärer Prof. Dr. Lumir Krejci Zürich, 2016 Table of contents Table of contents Table of contents ..................................................................................................................... 1 Zusammenfassung .................................................................................................................. 3 Summary .................................................................................................................................. 4 1 Introduction ........................................................................................................................... 5 1.1 DNA damage and repair ................................................................................................. 5 1.2 Replication and replication stress ................................................................................... 6 1.2.1 Eukaryotic DNA replication ...................................................................................... 6 1.2.2 Replication stress .................................................................................................... 8 1.2.3 Recovery of stalled replication forks ........................................................................ 9 1.3 Interstrand cross-link repair (ICL repair) ....................................................................... 10 1.3.1 Overview of the ICL repair pathway ....................................................................... 10 1.3.2 Types of ICL inducing agents ................................................................................ 11 1.3.3 Fanconi anemia proteins and pathway .................................................................. 12 1.3.4 Unhooking step in ICL repair ................................................................................. 15 1.3.5 Translesion polymerases and their roles in ICL repair .......................................... 18 1.3.6 Homologous recombination and ICL repair ........................................................... 20 1.3.7 Nucleotide excision repair and ICL repair .............................................................. 24 1.3.8 ICL repair in the Xenopus system .......................................................................... 25 1.3.9 Replication traverse of ICLs ................................................................................... 26 1.3.10 Replication-independent ICL repair ..................................................................... 27 1.3.11 ICL repair by glycosylases ................................................................................... 28 1.3.12 ICL repair in yeast ................................................................................................ 29 1.4 FEN1 ............................................................................................................................. 30 1.5 FAN1 ............................................................................................................................. 31 1.5.1 FAN1 discovery ..................................................................................................... 31 1.5.2 Roles of FAN1 outside of ICL repair ...................................................................... 32 1.5.3 FAN1 in Schizosaccharomyces pombe ................................................................. 32 1.5.4 FAN1 structure ....................................................................................................... 33 1.5.5 FAN1 in diseases ................................................................................................... 35 1.6 My aim ........................................................................................................................... 35 2 Results ................................................................................................................................. 37 2.1 FANCD2-associated Nuclease 1, but not Exonuclease 1 or Flap Endonuclease 1, is able to unhook DNA interstrand cross-links in vitro ............................................................ 37 2.2 Additional Observations ................................................................................................ 48 1 Table of contents 2.2.1 5' Flap cleavage activity in human TSCRE2 extracts ............................................ 48 2.2.2 FEN1 is responsible for almost all 5' flap cleavage in chicken cell extracts, but FAN1 activity becomes detectable upon overexpression ............................................... 49 2.2.3 FEN1 depletion sensitizes chicken cells to MMC and this effect can be rescued by FAN1 overexpression ..................................................................................................... 51 2.2.4 FAN1 protein levels in human cells are higher in the absence of FEN1 protein .... 52 3 Discussion .......................................................................................................................... 54 3.1 In vitro unhooking activity .............................................................................................. 54 3.2 In vivo implications of FAN1 unhooking activity ............................................................ 55 3.3 Evidence for FAN1 involvement in processes other than ICL unhooking ..................... 56 3.3.1 Replication fork recovery ....................................................................................... 57 3.3.2 Homologous recombination ................................................................................... 57 3.4 FAN1 and FEN1 ............................................................................................................ 58 3.5 FAN1 and SNM1A ......................................................................................................... 59 3.6 Potential parallel pathways to repair ICLs ..................................................................... 59 4 Conclusions and perspectives .......................................................................................... 61 5 Materials and Methods to section 2.2 ............................................................................... 62 5.1 Substrates for endonuclease assays ............................................................................ 62 5.2 Culturing of DT40 chicken cells ..................................................................................... 62 5.3 Culturing of TSCER2 cell lines and nuclear extract preparation ................................... 62 5.4 FAN1 overexpression in DT40 FEN1 KO cells ............................................................. 62 5.5 Preparation of whole cell extracts from DT40 cells ....................................................... 62 5.6 Nuclease assays with DT40 extracts ............................................................................ 63 5.7 Methylcellulose survival assay with DT40 cells ............................................................. 63 5.8 quantitative real-time (RT) PCR .................................................................................... 63 5.9 Antibodies ..................................................................................................................... 63 5.10 siRNAs ........................................................................................................................ 64 6 References .......................................................................................................................... 65 7 Acknowledgements ............................................................................................................ 76 8 Curriculum Vitae ................................................................................................................. 77 2 Zusammenfassung Zusammenfassung DNS-Reparaturmechanismen sind essentiell, um die genomische Stabilität aufrecht zu erhalten und dadurch auch um Krebs entgegenzuwirken. DNS Interstrangvernetzungen (ICLs), die beide DNS-Stränge kovalent miteinander verbinden, gehören zu den gefährlichsten DNS Schäden, da sie sowohl die Transkription als auch die Replikation blockieren. In höheren Eukaryoten werden ICLs auf eine komplizierte Art und Weise repariert und viele verschiedene DNS-Reparaturmechanismen sind daran beteiligt, wie Transläsionssynthese (TLS), Nukleotidexzisionsreparatur (NER) und homologe Rekombination (HR). Proteine der Fanconi-Anämie Komplementationsgruppe werden für die Koordination der Reparatur benötigt. Patienten mit Mutationen in Genen, welche für diese Proteine kodieren leiden an der schwerwiegenden, genetischen Krankheit Fanconi-Anämie. Der ICL-Reparaturweg beginnt mit dem Aushaken der Vernetzung
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