Genome Stability and Human Diseases (eBook)

Preface 6
References 7
Contents 8
Contributors 10
Coming Full Circle: Cyclin-Dependent Kinases as Anti-cancer Drug Targets 13
Introduction 14
Overview of Cell Cycle Control in Metazoans and Yeast 15
Cdk2 Becomes Dispensable 17
Whats Wrong with This Picture? 19
Closer to the Mark: Insights into CDK Function from Chemical Genetics 19
Cdk2 Back in the Saddle? 21
Conclusions and Perspectives 23
References 24
Core and Linker Histone Modifications Involved in the DNA Damage Response 28
Introduction 30
Histone Modifications 31
Mechanism of Action 32
Histone Modifications in DNA Repair 33
Core Histone Modifications in the Repair of DSBs 35
Phosphorylation of H2AX at S139 35
Phosphorylation of H2AX at Y142 36
Methylation of H3K79 38
Methylation of H4K20 39
Methylation of H3K9 39
Ubiquitylation of H2A and H2B 40
Acetylation of Histone H2AX 41
Acetylation of Histone H4 41
Acetylation of Histone H3 42
The Modification of Linker Histones During DNA Repair 43
Model for Integrated Role of Histone Modifications in Repair of DSBs 45
Aberrant Histone Modifications Cause Genome Instability and Disease 46
Histone Modifying Enzymes and Cancer 47
References 48
Chromatin Assembly and Signalling the End of DNA Repair Requires Acetylation of Histone H3 on Lysine 56 54
Introduction 55
Histone Acetylation in the DDR 56
H3K56ac in the DDR 58
H3K56ac in Human Cells 60
Conclusion 63
References 64
Structure and Function of Histone H2AX 66
Introduction 67
Chromatin Structure and Genome Stability 67
H2AX and DNA Repair 68
Structural Properties of H2AX 68
Definition of H2AX 69
H2AX Gene 70
H2AX Transcripts 72
H2AX Protein 72
H2AX Post-translational Modifications 75
H2AX Distribution in Chromatin 77
Combinatorial Potential in H2AX Distribution 77
Simulation of Random H2AX Inclusion 78
Functional Implications of H2AX Distribution 80
Possibility of Non-random H2AX Distribution 80
Functional Roles of H2AX 80
Initiation of H2AX Phosphorylation as a Reporter of DSB Events 81
Spreading of H2AX Phosphorylation as a Damage Signal Amplifier 81
H2AX and Chromatin Structural Remodelling 82
H2AX and Localisation of DSB Repair Proteins 83
H2AX and Maintenance of Proximity of Break Ends 84
H2AX and Complementary Damage Signalling via Ubiquitylation 85
Conclusion 85
References 85
The Initiation Step of Eukaryotic DNA Replication 90
Introduction 91
The Regulators of DNA Replication Initiation 92
Dpb11, Cut5 and TopBP1 92
Sld2 -- A New Player in the Initiation of DNA Replication 93
Sld3-- The Initiator of Initiation 94
The Replication Factor Cdc45 96
Discovery and Characterization of Cdc45 96
Expression of Cdc45 and Its Control 96
Dynamics of Cdc45 in the Cell 98
Interaction Partners of Cdc45 98
The Role of Cdc45 During DNA Replication 99
A Phosphorylation Switch for the Initiation of DNA Replication 99
GINS: An Evolutionarily Conserved Key Playerin DNA Replication 100
Identification of the GINS Complex 100
The Archaeal GINS Complex 102
Structural Studies on the GINS Complex 103
GINS in the Initiation and Elongation Phases of DNA Replication 105
Initiation and Checkpoint 107
A Role for Initiation Factors During Checkpoint Response 107
DNA Initiation Factors and Stalled DNA Replication Forks 108
Conclusions 108
References 109
Non-coding RNAs: New Players in the Field of Eukaryotic DNA Replication 116
Introduction 117
Non-coding RNAs in Eukaryotic DNA Replication 118
Y RNA 118
26T RNA 121
Structured G-Rich RNA 123
Conclusions 124
References 126
Function of TopBP1 in Genome Stability 130
Introduction 130
Role of TopBP1 in DNA Damage Signaling 132
Identification of TopBP1 as a Damage Response Protein 132
Involvement of ATM/ATR in TopBP1 Mediated Damage Response 132
Activation of ATR by TopBP1 134
Implications of TopBP1 in Response to DNA Double-Strand Breaks 136
ADP-Ribosylation of TopBP1 137
Regulation of TopBP1 Activity 137
TopBP1 in DNA Replication 139
A Role of TopBP1 During Mitosis and Meiosis 140
TopBP1 and Regulation of Transcription 141
Regulation of E2F1 141
SPBP and Ets1 Activation 142
Miz1 and UV Damage Response 143
Interaction with c-Abl 144
Hpv16 E2 144
Regulation of TopBP1 Gene Expression 145
TopBP1 and Cancer 145
Conclusions 146
References 147
Eukaryotic Single-Stranded DNA Binding Proteins: Central Factors in Genome Stability 153
General Overview 154
Replication Protein A 155
Physical Interactions of RPA with DNA 157
The RPA Complex and Its Binding to Proteins 159
The RPA Complex and DNA Replication 159
The RPA Complex in DNA Repair Processes -- Molecular Counting Capabilities 161
RPA Phosphorylation 163
An Alternative Form of Replication Protein A 164
Replication Protein A -- The Cancer Link 164
The Human ssDNA-Binding Protein hSSB1 165
Mitochondrial SSBs 166
Human mtSSB and p53 166
References 167
DNA Polymerases and Mutagenesis in Human Cancers 174
Genome Stability Control Mechanisms and the Replication Fork 174
DNA Repair and Mutagenesis 178
DNA Polymerases and Mutagenesis 184
Replicative Pols 184
DNA Repair Pols 185
TLS Pols 186
Concluding Remarks 189
References 190
DNA Polymerase , a Key Protein in Translesion Synthesis in Human Cells 198
Introduction 199
DNA Polymerase , a Member of the Y Family of Polymerases 200
Role of Pol in Bypass of Lesions Induced by Platinum-Based Chemotherapeutic Drugs 202
Role of Pol in Bypass of Other Lesions in DNA 204
Regulation of Pol Recruitment 204
Activation of DNA Damage Responses in Pol -Deficient Cells 207
Regulation of Pol Expression 209
Concluding Remarks 210
References 211
The Mitochondrial DNA Polymerase in Health and Disease 219
Introduction 219
Pol in mtDNA Replication 220
Pol in Mitochondrial DNA Repair 222
Mitochondrial Toxicity from Antiviral Inhibition of Pol 222
Disease Mutations in the POLG Gene 224
References 227
Centromeres: Assembling and Propagating Epigenetic Function 231
Introduction 232
Specifying the Centromere 233
Centromeric DNA 233
Evidence for Epigenetic Behavior at the Centromere 234
Chromatin and Centromere Determination 234
CENP-A, The Centromere Specific Histone 235
CENP-A Nucleosomes 236
Organization of CENP-A Within Centromeres 236
Assembly of CENP-A Chromatin and the Constitutive Centromere-Associated Network of Proteins 238
CENP-A Assembly in the Cell Cycle 238
Heritability and Dynamics of Centromeric Chromatin Proteins 239
Targeting CENP-A to the Centromere 240
Assembly of the Constitutive Centromere-Associated Network, CCAN 242
Kinetochore Function, Assembly and Signaling in the Spindle Assembly Checkpoint 245
Kinetochore Structure and Function 246
The Core Microtubule-Attachment Site -- KMN Network 247
Controlling Dynamics of Kinetochore-Microtubule Attachment and Chromosome Movement 247
Spindle Assembly Checkpoint (SAC): Maintaining Fidelity of Chromosome Segregation 248
Summary 250
References 251
Nucleotide Excision Repair in Higher Eukaryotes: Mechanism of Primary Damage Recognition in Global Genome Repair 258
Introduction 259
The Order of Protein Assembly in GG-NER 260
Advantages of Sequential Assembly of the Repair Complex 262
Models of DNA Damage Recognition 262
XPC-hHR23B Complex as a Potential Sensor of Helix Distortion 265
Role of UV-DDB in DNA Damage Recognition 266
Roles of XPA and RPA in NER 267
Photoreactive DNA Intermediates as a Tool to Study NER Assembly 269
Verification of Photoreactive dNMP Analogues as NER Substrates 269
Crosslinking of XPC-hHR23B to Photoreactive DNA is Moderated by XPA and RPA 272
Undamaged Strands are Strongly Required for XPC-hHR23B Crosslinking to Damaged DNA Duplexes 273
Localization of NER Factors on Undamaged Strand of Damaged DNA Duplex 275
References 278
Nonhomologous DNA End Joining (NHEJ) and Chromosomal Translocations in Humans 285
Frequency and Causes of Double-Strand Breaks 287
Vertebrate Nonhomologous DNA End Joining 288
The DNA Ligase IV Complex 289
The DNA Polymerases of NHEJ 290
Artemis, DNA-PKcs and the Nuclease of NHEJ 291
Concluding Comments on Vertebrate NHEJ 292
Chromosomal Translocations 292
Types of Translocations and Relation to Cancer 292
NHEJ in Chromosomal Structural Changes 293
Causes of DSBs That Initiate Translocations 293
Mistakes of V(D)J Recombination 295
Sequential Action by AID and the RAG Complex 295
Source of Single-Strandedness at Sites where AID Acts on meC Sites 298
Concluding Comments 298
References 299
Fluorescence-Based Quantification of Pathway-Specific DNA Double-Strand Break Repair Activities: A Powerful Method for the Analysis of Genome Destabilizing Mechanisms 303
Background 303
Implications of Pathway-Specific DNA Double-Strand Break Repair Activities 303
Fluorescence-Based DSB Repair Analysis 305
Materials 307
Solutions and Reagents 307
Equipment 308
Protocols 308
Transfection of MEFs with Repair Assay Plasmids 308
Co-transfection of MEFs with siRNA and Repair Assay Plasmids 309
Processing of Cells for Flow Cytometric Quantification of EGFP-Positive Cells 309
Processing Cells for Cell Cycle Analysis in 96-Well Plate 310
General Notes 311
References 311
Apoptosis: A Way to Maintain Healthy Individuals 313
Introduction 314
Apoptosis: General Considerations 314
Apoptosis During Embryogenesis and Development 316
Developmental Apoptosis and Model Organisms 316
Apoptosis and Germ Cells 317
Apoptosis in Neurons and Lymphocytes 318
Apoptosis and DNA Damage 319
Apoptosis Regulators in Response to DNA Damage 319
Apoptosis and Aneuploidy 320
Concluding Remarks 323
References 324
The Use of Transgenic Mice in Cancer and Genome StabilityResearch 330
Introduction 331
Evolution of Mouse Models of Cancer 332
Spontaneous and Carcinogen-Induced Cancers in Mice 332
Xenograft Models 332
Transgenic Mice 333
Production of Transgenic Mice 333
Pronuclear Injection 333
Genetically Engineered Mouse Embryonic Stem Cells 334
Types of Transgenic Mice 334
Traditional Transgenic Mouse Models 334
Conditional Transgenic Mouse Models 336
Uses of Transgenic Mice in Cancer Research 336
Mouse Models of Metastasis and Tissue Invasion 336
Mouse Models of Angiogenesis 337
Transgenic Mice Models in the Development of Cancer Therapeutics 337
Uses of Transgenic Mice in Genome Stability Research 338
Outlook 339
References 340
Index 342