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Chapter 7 Replication Checkpoint Dependent Rad4 Interactions A University of Sussex DPhil thesis Available online via Sussex Research Online: http://sro.sussex.ac.uk/ This thesis is protected by copyright which belongs to the author. This thesis cannot be reproduced or quoted extensively from without first obtaining permission in writing from the Author The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the Author When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given Please visit Sussex Research Online for more information and further details Protein-Protein Interactions Underlying Damage Checkpoint Activation in S. pombe Christopher Wardlaw Submitted for the Degree of Doctor of Philosophy University of Sussex September 2013 II Declaration I hereby declare that this thesis has not been and will not be, submitted in whole or in part to another University for the award of any other degree. Signature:……………………………………… III Acknowledgements I would like to thank Tony Carr for the opportunity to undertake my PhD in his laboratory, and for his great supervision, help and advice throughout the process. I would also like to thank Jo Murray for her helpful input, ideas and discussion, and thank my second supervisor Alessandro Bianchi. Many thanks to all of the Carr and Murray lab members, past and present, for their help and input over the past four years, and for creating such a wonderful working environment. Special thanks to Valerie Garcia for her supervision and support, and Adam Watson for his never ending technical help and patience. I am also very thankful to Stephanie Schalbetter and Yasu Daigaku for their advice and discussion, Chieh-Ju Lee for her microscope expertise, Charly Chawan for alignments and project ideas and Hung Quang Dang for his fusion PCR help. Many thanks to Su-Jiun Lin for her advice regarding the AAD project at the beginning of my PhD. Thank you to our collaborators Li Lin Du, Laurence Pearl and Tony Oliver for all of their contribution, discussion and work, and for including me in such a great project. I would like to thank the entire Brighton Bunch for their great friendship and support during my PhD, and for making it a very enjoyable time. Special thanks to Ann-Sophie, Carol, Diana, Iga, Sophie, Marcel, Michal, Ted and Yari. Thanks to all of my other friends from Brighton, London, Manchester and York for their support and continued friendship. A big thank you to all of my family, particularly my parents for everything they have done for me during my PhD and before. I would never have made it to this stage without all of the above people, thank you! IV UNIVERSITY OF SUSSEX Christopher Wardlaw A thesis submitted for the degree of Doctor of Philosophy Protein-Protein Interactions Underlying Damage Checkpoint Activation in S. pombe DNA damage can lead to the accumulation of mutations and diseases such as cancer. It is therefore integral for cells to identify this damaged DNA and promote its repair. To carry out this function eukaryotic cells have evolved signal transduction pathways known as the DNA structure checkpoints. Much of the molecular mechanism underlying these pathways is still far from understood. The work in this thesis uses the model organism Schizosaccharomyces pombe to investigate these mechanisms, with a particular focus on the TopBP1 homolog Rad4. TopBP1 plays an essential scaffolding role in the initiation of DNA replication, but is also a key protein in the DNA structure checkpoints. It has previously been shown in metazoans and budding yeast to stimulate the kinase activity of ATR, via its ATR Activation Domain (AAD), an early event in checkpoint activation. The work presented in here, along with initial work carried by previous members of the Carr Laboratory; Su-Jiun Lin and Valerie Garcia, shows that the Rad4TopBP1 AAD acts in a chromatin dependent pathway to amplify the checkpoint signal in G1/S-phase, where DNA resection is limited. A second AAD is also identified in the checkpoint clamp protein Rad9, which acts redundantly with the Rad4 AAD. As well as its AAD function, Rad4 also plays a scaffolding role in the DNA structure checkpoint pathways. The work in this thesis, in collaboration with the Laurence Pearl and Li Lin Du laboratories, identifies the molecular mechanism of the interaction between Rad4 and the mediator protein Crb253BP1. It is shown that sequential phosphorylation of Crb2 by Cdc2CDK is required for the interaction with BRCT domains 1 and 2 of Rad4 and checkpoint activation. It is also shown that Rad4 most likely does not interact with Mrc1 or Slx4 in the S. pombe checkpoint pathways. V Contents CHAPTER 1-INTRODUCTION ..................................................................... 1 1.0 General Introduction .................................................................................................. 1 1.1 Schizosaccharomyces pombe; an overview ................................................................ 1 1.2 The Cell Cycle .............................................................................................................. 3 1.2.1 Cell Cycle Overview ........................................................................................ 3 1.2.2 Regulation of Cell Cycle Progression .............................................................. 5 1.3 The DNA Damage and Replication Checkpoints ......................................................... 7 1.3.1 Overview of the DNA Damage and Replication Checkpoints ........................ 7 1.3.2 The Phosphatidylinositol (PI) 3 Kinase like Kinases (PIKKs) ......................... 11 1.4 The ATM/Tel1 Checkpoint ........................................................................................ 15 1.4.1 ATM Background .......................................................................................... 15 1.4.2 The Activation of ATM .................................................................................. 16 1.4.3 Mediators and the ATM Checkpoint Pathway ............................................. 21 1.4.4 Downstream Cell Cycle targets of the ATM pathway .................................. 23 1.4.5 Role of the Yeast ATM Homolog, Tel1, in Checkpoint activation ................ 23 1.5 ATR/Rad3/Mec1 Pathway of Checkpoint Activation ................................................ 25 1.5.1 ATR/Rad3/Mec1 Background ....................................................................... 25 1.5.2 The Formation of ssDNA .............................................................................. 27 1.5.3 Recruitment of ATR to ssDNA ...................................................................... 30 1.5.4 The DNA Damage Sensor 9-1-1 is Required for the ATR Dependent Checkpoint ............................................................................................................. 31 1.5.5 The Activation of the ATR Checkpoint Pathway .......................................... 32 1.5.6 ATR and the Replication Checkpoint ............................................................ 37 1.5.7 ATR Checkpoint Maintenance and Amplification ........................................ 40 1.6 TopBP1; a Review ..................................................................................................... 41 1.6.1 Identification of TopBP1 and its Homologs .................................................. 42 1.6.2 TopBP1 Domain Architecture ....................................................................... 42 1.6.3 TopBP1 as a Scaffold in the DNA Checkpoints. ............................................ 44 VI 1.6.4 TopBP1 Dependent ATR Activation .............................................................. 48 1.6.5 TopBP1 and DNA Repair ............................................................................... 51 1.6.6 TopBP1 and the Initiation of DNA Replication ............................................. 52 1.6.7 TopBP1 and the Regulation of Transcription. .............................................. 56 1.6.8 TopBP1 and Disease ..................................................................................... 59 1.7 Aims of this Work...................................................................................................... 60 CHAPTER 2-MATERIALS AND METHODS ................................................ 62 2.1 Media ........................................................................................................................ 62 2.1.1 Yeast Media .................................................................................................. 62 2.1.2 Bacteria Media ............................................................................................. 63 2.1.3 Chemicals and Drugs used for Selection ...................................................... 63 2.2 General Molecular Techniques ................................................................................. 64 2.2.1 DNA Restriction Digests ............................................................................... 64 2.2.2 Plasmid DNA Ligations .................................................................................. 64 2.2.3 PCR for Molecular Cloning ........................................................................... 64 2.2.4 Fusion PCR .................................................................................................... 65 2.2.5 Site Directed Mutagenesis (SDM) ................................................................ 65 2.2.6
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