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Structure and Expression of the Mouse Dna Ligase I Gene

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Structure and Expression of the Mouse Dna Ligase I Gene STRUCTURE AND EXPRESSION OF THE MOUSE DNA LIGASE I GENE Joanne Kirsty Jessop A Thesis Presented for the Degree of Ph.D. Institute of Cell and Molecular Biology The University of Edinburgh February 1995 DECLARATION The composition of this thesis and the work presented in it are my own, unless otherwise stated. The experiments were designed in collaboration with my supervisor Dr. David W. Melton. Joanne Kirsty Jessop February 1995 CONTENTS .Abstract 1 Acknowledgements 3 Abbreviations 4 Chapter 1 Introduction 7 1.1 Foreword 8 1.2 DNA Repair 8 1.2.1 Direct Reversal of DNA Damage 11 1.2.2 Base Excision Repair 12 1.2.3 Nucleotide Excision Repair 12 1.2.4 Post-Replication Repair 12 1.3 Human Diseases Arising From Defective DNA Repair 17 1.3.1 Xeroderma Pigmentosum 17 1.3.2 Cockaynes Syndrome 19 1.3.3 Ataxia Telangiectasia 20 1.3.4 Fanconi Anaemia 20 1.3.5 Blooms Syndrome 21 1.3.6 The DNA Ligase I-Deficient Cell Line 46BR 24 1.4 Mammalian DNA Ligases 28 1.4.1 DNA LigaseI 31 1.4.1.1 Substrate Specificity of DNA Ligase I 31 1.4.1.2 The Human DNA Ligase I Gene 31 1.4.1.3 DNA Ligase I Structure 32 1.4.1.4 Regulation of DNA Ligase I Activity 33 1.4.1.5 Intracellular Location and Function of DNA Ligase I 34 1.4.2 DNA Ligasell 35 1.4.3 DNA Ligase III 36 1.5 Gene Targeting and the Creation of Mouse Models for Human Genetic Disease 37 1.6 Project Aim 43 Chapter 2 Materials and Methods 47 2A Materials 48 2A.1 Suppliers of Laboratory Reagents 48 111 2A. 1.1 Restriction Endonucleases and other DNA/RNA Modifying Enzymes 48 2A.1.2 Standard Laboratory Reagents 48 2A.1.3 Bacterial Media Reagents 48 2A. 1.4 Mammalian Cell Culture Reagents 48 2A.1.5 Radioactive Reagents 48 2A.1.6 Antibiotics 48 2A.1.7 Antisera 49 2A.1.8 Human DNA Ligase I cDNA 49 2A.1.9 Oligonucleotides 49 0 2A.2 Media 50 2A.2.1 Bacterial Media 50 2A.2.2 Mammalian Tissue Culture Media 51 2A.3 Bacterial Strains 52 2A.4 Mammalian Cell Lines 53 2A.5 Plasmids and Cloning Vectors 53 2A.6 Buffers 54 Methods 55 2B.1 Bacterial Culture 55 2B. 1.1 Growth of Escherichia coli 55 2B.1.2 Storage of E. coli 55 2B. 1.3 Transformation of E. coil 55 2B.2 Nucleic Acid Isolation 56 213.2.1 Small Scale Preparation of Plasmid DNA 56 213.2.2 Large Scale Preparation of Plasmid DNA 56 2B.2.3 Preparation of Genomic DNA from Mammalian Cells 57 2B.2.4 Preparation of RNA from Mammalian Cells 57 2B.2.5 Small-Scale Preparation of Bacteriophage M13 Replicative Form DNA 58 2B.2.6 Preparation of Single-Stranded Bacteriophage M13 DNA 59 2B.2.7 Preparation of Bacteriophage ?s DNA 59 2B.3 Quantification of Nucleic Acids 60 2B.3.1 Estimation of DNA Concentration 60 2B.3.2 Estimation of RNA Concentration 60 iv 2B.4 DNA Manipulation 60 2B.4.1 Purification 60 2B.4.2 Restriction of DNA with Endonucleases 61 2B.4.3 Dephosphorylation 61 2B.4.4 Filling-In Recessed 3' DNA Termini 61 2B.4.5 Ligation 61 2B.4.6 Addition of Linkers 62 2B.5 Electrophoresis of Nucleic Acids 62 2B.5.1 Electrophoresis of DNA in Agarose Gels 62 2B.5.2 Electrophoresis of RNA in Agarose Gels 62 2B.5.3 Recovery of DNA from Agarose Gels 62 2B.6 Transfer of Nucleic Acids from Agarose Gels to Membranes 63 2B.6.1 DNA Transfer 63 2B.6.2 RNA Transfer 64 2B.7 Nucleic Acid Hybridisation 64 2B.7.1 Labelling DNA by Random Priming with Hexadeoxy-ribonucleotide Primers 64 2B.7.2 Separation of Unincorporated Nucleotides from Labelled DNA 64 2B.7.3 Hybridisation 65 2B.7.4 Autoradiography 65 2B.7.5 Stripping Probes from Filters 65 2B.8 cDNA and Genomic Libraries 66 2B.8.1 cDNA Synthesis 66 2B.8.2 Preparation of Packaging Extracts for in vitro Packaging of Bacteriophage ? 66 2B.8.3 Library Construction 67 2B.8.4 Plating out Bacteriophage ? to generate Plaques 67 2B.8.5 Probing Libraries 68 2B.9 DNA Sequencing 68 2B.9.1 Templates for Sequencing 68 2B.9.2 Annealing Primer to Template DNA 68 2B.9.3 Sequencing 69 2B.9.4 Electrophoresis of Sequenced DNA 70 2B.10 Amplification of DNA by the Polymerase Chain Reaction 70 V 2B.10.1 Polymerase Chain Reaction Experiments 70 2B. 10.2 Screening Colonies Using the Polymerase Chain Reaction 71 2B. 11 Cell Culture 71 2B.11. 1 Growth of Mammalian Cells 71 2B.1 1.2 Electroporation 71 2B.1 1.3 Calcium Phosphate/DNA Precipitation and Colony-Forming Assay 71 2B.11.4 Ethyl Methane Suiphonate Survival Assay 72 2B. 11.5 3-Aminobenzamide Survival Assay 72 2B.12 Protein Methods 72 2B. 12.1 Expression of Proteins in E. co/i 72 2B. 12.2 Preparation of Protein Extracts from Mammalian Cells 73 2B. 12.3 Electrophoresis of Proteins 73 2B. 12.4 Transfer of Proteins From Polyacrylamide Gels to Membranes 74 2B. 12.5 Probing Protein Blots with Antibodies 74 Chapter 3 Phenotypic Correction of the Human Cell Line 46BR 75 3.1 Construction of a Vector Expressing Human DNA Ligase I cDNA 76 3.2 Transfection 79 3.3 Detection of DNA Ligase I Transfectants 79 3.4 Expression of Wild-Type Human DNA Ligase I cDNA 82 3.5 Correction of Ethyl Methane Suiphonate Sensitivity in 46BR 85 3.6 Correction of 3-Aminobenzamide Sensitivity in 46BR 88 Chapter 4 Mouse DNA Ligase I eDNA 91 4.1 Cloning of Mouse DNA Ligase I cDNA 92 4.2 The Ligase Clones Encode Mouse DNA Ligase I 92 4.3 Restriction Analysis of Mouse DNA Ligase I cDNA 97 4.4 Sequencing of Mouse DNA Ligase I cDNA 97 Chapter 5 Expression of Mouse DNA Ligase I cDNA 113 5.1 Introduction of a Marker into Mouse DNA Ligase I 114 5.2 Expression of Flag-Ligase in E. coli 119 5.3 Expression of Hag-Ligase in 46BR Under Control of the vi Phosphoglycerate Kinase Promoter 124 5.4 Expression of Flag-Ligase in 46BR Under Control of the Human 13-Actin Promoter 133 Chapter 6 Mouse DNA Ligase I Gene Structure 138 6.1 Southern Hybridisation Analysis 139 6.2 Mouse DNA Ligase I Gene Map 155 Chapter 7 Cloning of a Genomic Fragment Containing the Arginine to Tryptophan Mutation Site 163 7.1 Cloning of Mouse DNA Ligase I Genomic Fragments 164 7.2 Restriction Mapping and Intron/Exon Structure of the 13.5kb EcoRT Genomic Fragment 167 7.3 Identification of the Arginine to Tryptophan Mutation Site in the 13.5kb Mouse EcoRI Genomic Fragment 177 Chapter 8 Gene Targeting at the Mouse DNA Ligase I Locus 182 8.1 Targeting Vector Construction 184 8.2 Targeting the Mouse DNA Ligase I Gene 188 8.3 Mouse Models for DNA Ligase I Deficiency 199 Chapter 9 Discussion 201 References 211 ABSTRACT 46BR is a cell line derived from a patient suffering from immunodeficiency, growth retardation and sunlight sensitivity. DNA replication and repair is affected in 46BR cells which are sensitive to the alkylating agent ethylmethane sulphonate and to 3-aminobenzamide. Aberrant DNA ligase I activity has been demonstrated in these cells, and mutations have been identified in both alleles of the DNA ligase I gene. The first mutation specifies a change of glutamate 566 to lysine, a charge alteration which renders the enzyme completely inactive and which could be lethal in a homozygote. The second mutation is a change of arginine 771 to tryptophan which leaves the enzyme with some residual activity. The transformed derivative of 46BR is either hemizygous or homozygous for this second mutation, so cells expressing only this mutant form of DNA ligase I are still viable. This mutation is therefore a good candidate for introduction into the mouse gene in order to create a mouse model for DNA ligase I deficiency. Prior to making a mouse model it was necessary to show that the mutations at the DNA ligase I locus in 46BR give rise to the aberrant cellular phenotype. To do this, wild-type human DNA ligase I cDNA was introduced into 46BR in an expression vector. Selection for transformants was provided by a bacterial neomycin phosphotransferase gene. Transformants were assayed for survival in ethylmethane sulphonate and 3-aminobenzamide, which demonstrated a correlation between the presence of wild-type DNA ligase I sequences within the cells and rescue of sensitivity to these agents. Wild-type human DNA ligase I cDNA therefore complements the defect in 46BR. A 3.2kb mouse DNA ligase I cDNA clone was obtained by probing a mouse embryonic stem cell cDNA library with full-length human DNA ligase I cDNA. The cDNA clone is amost complete, but lacks sequence from the 3' end, including the polyadenylation signal. At the amino acid level, homology between human and I mouse DNA ligse I is extremely high, with most sequence variation in the N- terminal regulatory domain. In the C-terminal catalytic domain the amino acid sequences are virtually the same, with identical amino acids in the same positions at which the two mutations were identified in 46BR. The mouse cDNA clone was used to analyse the mouse DNA ligase I gene structure by hybridisation to mouse genomic DNA.
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