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Biochemical Analysis Of Yeast Pre-Replicative Complex Assembly Katie Anne Halford Cancer Research UK And University College London Submitted for the degree of Doctor of Philosophy 2003 ProQuest Number: 10010077 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest 10010077 Published by ProQuest LLC(2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 Abstract The boundaries of replication research would be greatly extended by the establishment of an origin-dependent in vitro DNA replication system. Saccharomyces cerevisiae is a prime candidate from which to develop such a system, as it initiates replication from specific, well conserved, short origin sequences. The first step towards producing a yeast cell free replication system was the development of a method to assemble pre-replicative complexes (pre-RCs) onto short repeat sequences of the origin ARSl, immobilised on Dynabeads. I have extended this research to use plasmids with ARSl origins. The plasmids are competent for pre-RC assembly (ORC, Cdc6p, MCM) and this is specific for origin sequences with an A element, the element known to be essential for replicationin vivo. Formation of the complexes is also time dependent and once the MCM complex has been loaded, it can stay associated even in the absence of the MCM loader Cdc6p. This is in accordance with in vivo data and suggests that the system will be useful to elucidate in vivo mechanisms of replication. I have also explored the kinetics of pre-RC assembly using different nucleotides and competitor ARSl DNA. Different steps in pre-RC assembly have different nucleotide requirements and the loading of substantial levels of the MCM complex requires the hydrolysis of ATP. Loaded MCM complexes are stable on DNA, even in the presence of competitor ARS1 plasmid. I have further investigated the nucleotide requirements using mutants of Cdc6p with changes in an ATP interaction region. Recombinant Cdc6p was produced in E. coli and purified by means of a poly-histidine tag. Cdc6p or mutant cdc6p was also expressed as a second copy in yeast cells and extracts were produced containing these proteins. Experiments with rCdc6p or the yeast extracts produced comparable results. I have successfully established in vitro loading of pre-replicative complexes at ARS 1 origins on plasmid DNA and begun to investigate the requirements of this system. Table of Contents Title.............................................................................................................................................1 A bstract ..................................................................................................................................... 2 Table of Contents..................................................................................................................... 3 List of Figures...........................................................................................................................8 Abbreviations ..........................................................................................................................10 Acknowledgements ................................................................................................................ 11 Chapter 1 Assembly of Pre-Replicative Complexes.........................................................12 1.1 Introduction............................................................................................................. 13 1.2 Replication Origins and Initiator proteins............................................................ 13 The Replicon Model and Initiation of DNA Replication inEscherichia coli .............13 SV40 and the initiation of DNA Replication.................................................................14 Initiation of DNA replication atSaccharomyces cerevisiae origins ............................ 15 Initiation of DNA replication atSchizosaccharomyces pombe origins........................19 Initiation of DNA replication in Higher Eukaryotes....................................................21 1.3 The Formation of Pre-Replicative Complexes at origins....................................24 1 The association o f ORC with origins ......................................................................... 24 ORC binds origins throughout the cell cycle................................................................ 24 ORC and the role of ATP...............................................................................................25 2 Cdc6 and Cdtl proteins are recruited to origins .......................................................27 3 Loading o f the MCM2-7 complex .............................................................................. 28 4 The Timing o f initiation from replication origins ......................................................32 5 Pre-RC maturation into pre-initiation Complex (pre-IC) ........................................34 6 The initiation o f DNA replication and the roles o f two protein kinases ...................35 Cyclin Dependent Kinase...............................................................................................35 Cdc7-Dbf4 kinase ............................................................................................................37 Targets of CDK and Cdc7 for the Initiation of DNA Replication.............................. 39 1.4 Once per Cell Cycle DNA Replication................................................................. 40 Phosphorylation of ORC................................................................................................43 Cdc6p, Regulation of abundance and activity .............................................................44 CDTlp, changes in abundance and negative regulation ............................................. 45 MCM2-7, subcellular localisation and inhibition of helicase activity....................... 46 1.5 Conclusions............................................................................................................. 47 Chapter 2 Materials and Methods...................................................................................... 49 2.1 Yeast Strains.............................................................................................................50 2.1.1 Yeast Strains....................................................................................................50 2.1.2 Medium ............................................................................................................50 2.1.3 Storage.............................................................................................................50 2.2 Bacterial Strains...................................................................................................... 51 2.2.1 Bacterial Strains.............................................................................................. 51 2.2.2 Medium ............................................................................................................52 2.2.3 Storage.............................................................................................................52 2.2.4 Transformation ofE. coli ............................................................................... 53 2.3 Production of Yeast Extracts...................................................................................54 2.3.1 Extracts with Cdc6p ........................................................................................54 2.3.2 Extracts free of Cdc6p .....................................................................................55 2.3.3 Extracts for testing mutations in CDC6........................................................ 56 2.3.4 Ammonium sulphate cut extracts..................................................................56 2.4 The Loading Assay..................................................................................................57 2.4.1 Production of ARS 1 beads.............................................................................57 2.4.2 The Loading Assay with extracts.................................................................. 59 2.4.3 The Loading Assay with semi-purified ORC............................................... 61 2.4.4 The Loading Assay with competitor DN A...................................................61 2.5 Purification of ORC.................................................................................................61 2.6 Purification of rCdc6 ...............................................................................................63 2.6.1 The strains for rCdc6p expression .................................................................63 2.6.2 Induction of Cdc6p .........................................................................................64 2.6.3 Cell Lysis......................................................................................................... 65 2.6.4 Buffers for Cdc6p purification .....................................................................
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