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Nucleosome Assembly and Histone H3 Methylation During Dna Replication

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Nucleosome Assembly and Histone H3 Methylation During Dna Replication NUCLEOSOME ASSEMBLY AND HISTONE H3 METHYLATION DURING DNA REPLICATION TxrwTom Rolef Ben-Shahar A thesis submitted for the degree of Ph.D. at The University of London July, 2004 Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms, Hertfordshire, EN6 3LD and University College London, Gower Street, London, WC1 6BT ProQuest Number: 10014844 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 10014844 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 ACKNOWLEDGEMENTS To begin with, I would like to acknowledge my mistakes, scientific and others. Hopefully the future will show that I have learned what I could from them. My work has been funded by Cancer Research UK and with a B’NAI BRITH scholarship, and for that I am grateful. I would like to thank Alain Verreault for his supervision, for our scientific conversations and for allowing me to make my own mistakes. My gratitude is given to Munah Abdul-Rauf for being a colleague, collaborator, friend and sister; to Michal Goldberg, Niki Hawks and to other friends and colleagues in Clare-Hall. I would also like to thank the people at the service units here at Clare Hall, and in Lincoln Inn Fields. A special thanks to Yossi Gruenbaum for having faith in me, and to Wendy for her support. My heart is always with Anath who gave me perspective in recent years. Last but not least to Asaf, I thank you for everything. ABSTRACT Nucleosome assembly is essential for viability and entails a controlled mechanism by which newly synthesised histones are specifically deposited onto replicating DNA by assembly factors such as Chromatin Assembly Factor 1 (CAF1). CAF1 binds directly to Proliferating Cell Nuclear Antigen (PCNA), a processivity factor for DNA replication and repair, and this interaction is necessary to target its 'cargo', histones H3/H4, to newly synthesised DNA. Chapter 3 in this work presents the characterisation of the interaction between CAF1 and PCNA. This interaction is surprisingly complex, involving two distinct consensus PCNA binding motifs in the p150 subunit of CAF1, which are functionally distinct. Similar motifs are found in many other proteins involved in cell cycle control, DNA replication, DNA modification and various forms of DNA repair. I found some specificity in binding to PCNA, underscoring the existence of a regulatory mechanism which ensures that CAF1 and other PCNA-binding proteins act in a well-orchestrated manner at the replication fork. In Chapters 4 and 5, I employed two different techniques to identify other factors involved in chromatin assembly in human cells and in yeast. Micro­ array analysis in budding yeast revealed global changes in gene expression in chromatin assembly factors null mutants. Through this screen I identified Periodic Tryptophan Protein 1 (Pwp1) as a putative chromatin assembly factor. The faithful replication and transfer of hereditary material to daughter cells includes not only accurate duplication of the parental DNA and chromosome architecture, but also the propagation of an epigenetic ‘signature’. DNA replication offers a window of opportunity for cells to duplicate or modify the epigenetic state. For instance, DNA méthylation is propagated during replication by the binding of the maintenance DNA Methyltransferase DNMT1 to PCNA. In Chapter 6, I used the budding yeast to examine a possible role for PCNA in directing the specialised histone H3 lysine 4 methyltransferase Set1 to newly assembled chromatin. ABBREVIATIONS 2D Two Dimensional A Deletion mutant Ab Antibody ECL Enhanced Ada Adenine Chemiluminescence AC F ATP utilising Chromatin EDTA Ethylene diamine tetra- assembly and remodelling acetic acid Factor EGFP Enhanced GFP ALB Agarose Loading Buffer ERFP Enhanced RFP Amp Ampicillin ExPasy Expert Protein Analysis Apn2 AP nuclease 2 System APS Ammonium persulphate FACT Facilitates Chromatin Transcription ARS Autonomously Replicating Sequence FBS Foetal Bovine Serum Asf1 Anti-silencing factor 1 FEN1 Flap Endonuclease 1 ATP Adenosine 5'-triphosphate FOA 5-Fluoro-orotic acid BER Base Excision Repair FPLC Fast performance liquid chromatography BLAST Basic Local Alignment GADD Growth Arrest and DNA Search Tool 45 Damage induced protein 45 Bp Base pairs GAL Galactose BrDU Bromodeoxyuridine GEB GST Elution Buffer BSA Bovine Serum Albumin GFP Green Fluorescent Protein pME p-Mercaptoethanol GO Gene Ontology CAC Chromatin Assembly GST Glutathione-S-transferase Complex HA Haemagglutinin CAF1 Chromatin Assembly Factor 1 HAST Hda1-Affected Subtelomeric Cdc Cell division cycle domains CDK Cyclin Dependent Kinase HAT Histone Acetyltransferase CHRAC Chromatin Accessibility HD1 Histone Deacetylase 1 Complex HD AC Histone Deacetylase D Dimérisation Domain HEPES N-(2-Hydroxyethyl) DDK Dbf4-dependent kinase piperazine-N'-(2- dHgO Distilled water ethanesulfonic acid) DEPC Diethylpyrocarbonate Hir Histone gene regulation DMSO Dimethyl sulphoxide HLB His Lysis Buffer DNA Deoxyribonucleic Acid HML Homothalic Mating type loci L DNA- DNA-Protein Kinase HMR Homothalic Mating type loci R PK HMT Histone Methyltransferase DNMT DNA Methyltransferase HP1 Heterochromatin Protein 1 dNTP Deoxy N-5'-triphosphate HRP Horseradish Preoxidase HU Hydroxyurea Ore Origin Recognition Complex IDCL Inter-Domain Connecting ORF Open Reading Frame Loop pAb Polyclonal antibody IPTG Isopropyl-p-D- PAS Protein A Sepharose thiogalactopyranoside PBM PCNA Binding Motif ISWI Imitation Switch PBP PCNA Binding Protein Kan Kanamycin PBS Phosphate Buffered Saline Kb Kilo base PBS-T PBS + 1% Tween20 Kd Dissociation constant PBS- PBS-T + 5% BSA kDa Kilo Dalton TB LB L Broth PBS- PBS-T + 5% skimmed Milk Leu Leucine TM Lig1 DNA ligase 1 PCR Polymerase Chain Reaction mAb Monoclonal antibody PCNA Proliferating Cell Nuclear MBD Methyl-DNA binding Antigen MCM Micro-Chromosome PEI Polyethyleneimine Maintenance pi Isoelectric point MeCP Methyi-CpG binding Protein PI Protease Inhibitors MIPS Munich Information Centre PMSF Phenylmethylsulfonyl for Protein Sequences Fluoride MLB Mammalian Lysis Buffer Pol DNA polymerase MLH Mut L homologue PP1 Protein Phosphatase 1 MMR Mismatch Repair PPase Protein Phosphatase MMS Methylmethanesulfonate Pre-IC Pre-Initiation Complex MO! Multiplicity Of Infection Pre-RC Pre-Replication Complex MSN Mut S homologue PVP Position-effect Variegation MyD Myeloid Differentiation PWP1 Periodic Tryptophan Protein 1 118 primary response gene 118 RAS Retrovirus-Associated DNA NAP Nucleosome Assembly Sequences Protein Rb Retinoblastoma NCBI National Centre for RCAF Replication-Coupling Biotechnology Information Assembly Factor NER Nucleotide Excision Repair rDNA Ribosomal DNA NGS Normal Goat Serum RFC Replication Factor C nt nucleotides RFP Red Fluorescent Protein NuRD Nucleosome Remodelling RNA Ribonucleic Acid histone Deacetylase RNase RNA nuclease HI complex HI NURF Nucleosome Remodelling RPA Replication Protein A Factor rpm Revolutions per minute OD Optic Diffraction rRNA Ribosomal RNA ON Overnight RT Room Temperature TE Tris EDTA solution S100 Cytosolic Buffer TEMED N,N,N',N'- SAGE Serial Analysis of Gene T etramethylethylenediamine Expression Tm Melting Temperature Sas2 Something About Silencing 2 tRNA Transfer RNA SDM Site Directed Mutagenesis Tris Tris (hydroxymethyl) SDS- Sodium Dodecyl Sulphate- aminomethane PAGE Polyacrylamide Gel Trp Triptophan Electrophoresis TSA Trichostatin Set1 Su(var)3-9 - enhancer of TSS Transformation and Storage zeste - trithorax 1 Solution SGD Saccharomyces Genome Tween Polyoxyethylene (20) Database 20 sorbitan monolaurate Sir Silent information regulator Ubc9 Ubiquitin-like protein SUMO- sIRNA small interference RNA 1 conjugating enzyme Snf2/S Sucrose non-fermenting UNG2 Uracil N-Glycosylase 2 NF2H yeast homologue-like 2 Ura Uracil SSB Single Strand Break UV Ultraviolet SsDNA Single Stranded DNA WICH WSTF-ISWI Chromatin SUV39 Suppressor of Variegation remodelling complex H1 3-9 homologue 1 Xic X inactivation centre SV40 Simian Virus 40 Xist Inactive X specific transcript TAFII TATA-binding protein XPG Xeroderma Pigmentosum G Associated Factor II YNB Yeast Nitrogen Base I Antigen T Ag YPD Yeast Peptone Dextrose TOA Trichloroacetic Acid CONTENTS Page Acknowledgements ...................................................................................... 2 Abstract......................................................................................................... 3 Abbreviations ................................................................................................ 4 Contents ......................................................................................................... 7 Tables and figures ......................................................................................... 10 Chapter 1 - Introduction 1.1 Historical Perspective ....................................................................... 13 1.2 S phase and the eukaryotic cell cycle ............................................ 14 1.3 DNA replication ................................................................................
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