Contents

Back to Basics: Structure, Function, Evolution and

Application of Homing Endonucleases and Inteins 1

Marlene Belfort

1 Introduction: Back to Basics 1

2 What Is a Homing Endonuclease? 2

3 What Is an Intein? 4

4 Inteins and Homing Endonucleases as Molecular Mosaics. 4

5 Applications: "Turning Junk into Gold" 6

5.1 Site-Specific Group I Intron and Intein Endonucleases . . . 6

5.2 Gene Targeting by a Group II Intron RNP Complex 6

5.3 "Inteins ... Nature's Gift to the Protein Chemist" 8

6 The Message 9

References 10

Homing Endonucleases and the Yeast

Mitochondrial œ Locus - A Historical Perspective 11

Bernard Dujon

1 The First Years of Yeast Mitochondrial Genetics 11

2 Genetic and Molecular Characterization of the œ Locus .. 13

3 The Problems of Mitochondrial Intronic

Reading Frames and Their Products 17

4 Group I and Group II Introns, and RNA Splicing 18

5 First Clues to the Function of the

Translation Product of the œ Intronic Reading Frame ... 19

6 Expressing the œ Intron-Encoded Protein in a Heterologous System 21

7 The Unusual Enzymatic Properties of the

Group I Intron-Encoded Homing Endonuclease I-Scel... 22

8 The First Additional Homing Endonucleases

Discovered after I-Scel 23

9 Use of the I-Scel Endonuclease in Heterologous Systems 24

10 Epilogue 26

References 26

The LAGLIDADG Homing Endonuclease Family 33

Brett Chevalier, Raymond J. Monnat, Jr., Barry L. Stoddard

1 Introduction 33

2 Structures of LAGLIDADG Homing Endonucleases 35

3 Mechanisms of DNA Target

Site Recognition and Specificity 38

4 Mechanism of DNA Cleavage 41

References 45

HNH Endonucleases 49

Anthony H. Keeble, Mari'a J. Mate, Colin Kleanthous

1 Introduction 49

2 The HNH Family - A Tree of Three Branches 50

2.1 The HNH Consensus Sequence 50

2.2 The Cysteine-Containing HNH Motif (cysHNH) 51

2.3 The HNN Variant 52

2.4 The HNH Motif Is Part of the

Wider ppa-Me Superfamily of Endonucleases 52

3 Enzymology of HNH/ppa-Me Motif Endonucleases 54

3.1 The HNH/ppa-Me Motif Is Functionally Adaptable 54

3.2 Biochemical Properties 56

3.3 HNH/ppa-Me Enzymes Require Single

Divalent Cations for Activity 57

4 Structural Analysis of HNH

Endonuclease Mediated dsDNA Cleavage 58

4.1 Relaxation of Substrate Strain is Conserved in the

Cleavage Mechanisms of HNH/ppa-Me Enzymes 58

4.2 Metal Ion Coordination in the DNA-Bound Complex. ... 59

4.3 Mechanism of Mg2+-Dependent Cleavage by HNH Endonucleases and Why Zn2+

Does Not Support Catalytic Activity 60

5 Conclusions 62

References 63

GIY-YIG Homing Endonucleases - Beads on a String 67

Patrick Van Roey, Victoria Derbyshire

1 Introduction 67

2 Occurrence and Functions of GIY-YIG Enzymes 69

3 I-TevI as the Model GIY-YIG Enzyme:

Structure and Function 70

3.1 Catalytic Domain 71

3.2 DNA-Binding Domain 74

3.3 Flexible Linker and Distance Determination 75

3.4 I-TevI Endonuclease Is Bifunctional,

Also Serving As a Transcriptional Autorepressor 77

4 DNA-Binding Domain Diversity and Conserved Modules . 79 References 81

His-Cys Box Homing Endonucleases 85

Eric A. Galburt, Melissa S. Jurica

1 The His-Cys Box Family of Homing Endonucleases 86

2 Expression of His-Cys Box Homing

Endonucleases from Nuclear rDNA Transcripts 89

3 Structure, DNA Binding, and Catalytic Mechanism 91

3.1 Structure of a His-Cys Box Endonuclease: I-Ppol 91

3.2 DNA Binding and Recognition 93

3.3 Catalytic Mechanism 96

3.3.1 Catalytic Metal Coordination 96

3.3.2 Alignment and Activation of the Hydrolytic Water 97

3.3.3 Conformational Changes and

Transition State Stabilization 98

4 HNH and His-Cys Box Homing Endonucleases 99

References 100

Group I Introns and Their Maturases:

Uninvited, but Welcome, Guests 103

Mark G. Caprara, Richard B. Waring

1 Discovery of Self-Splicing Group I Introns 104

2 Genes Within Introns 106

3 How Many Maturases? 106

4 The Mechanism of Maturase-Assisted Splicing 107

5 Maturases Cooperate with Other Proteins for Splicing . .. 110

6 Maturases or DNA Endonucleases? 110

7 How Intertwined Are the DNA and RNA

Activities in Maturases? Ill

8 Evolution of Maturase Activity 113

9 Concluding Remarks 114

References 116

Group II Intron Homing Endonucleases: Ribonucleoprotein

Complexes with Programmable Target Specificity 121

Alan M. Lambowitz, Georg Mohr, Steven Zimmerly

1 Introduction 121

2 Group II Intron Structure 122

3 Catalytic Properties of the Intron RNA 122

4 Biochemical Activities of Group II

Intron-Encoded Proteins 125

5 Mobility Mechanisms 126

6 Binding of the IEP to the Intron RNA 128

7 DNA Target Site Recognition by Group II

Intron Homing Endonucleases 130

8 DNA Target Site Recognition by LLLtrB Intron RNPs. ... 132

9 Group II Introns as Gene-Targeting Vectors 135

References 141

Free-Standing Homing Endonucleases of T-Even Phage:

Freeloaders or Functionaries? 147

David R. Edgell

1 Introduction 147

2 When Is a Free-Standing Endonuclease a

Homing Endonuclease? 148

3 Intronless Homing and Marker Exclusion 149

4 The Recognition Sites of Free-Standing Endonucleases

Are Distinct from the Endonuclease Insertion Site 152

5 How Do Free-Standing Endonucleases

Prevent Cleavage of Their Host Genome? 153

6 The Separation of Cleavage and Insertion

Sites Influences Endonuclease Mobility 154

7 The Sporadic Distribution of

Free-Standing Homing Endonucleases 155

8 The "Function" of Free-Standing Endonucleases 156

9 The Diversity of Endonuclease Function 157

References 158

Function and Evolution of HO

and VDE Endonucleases in Fungi 161

James E. Haber, Kenneth H. Wolfe

1 Introduction 161

2 Mating-Type Genes in S. cerevisiae 162

3 HO-Induced MAT Switching in S. cerevisiae 163

4 Mechanism of MAT Switching 164

5 Donor Preference Associated with MAT Switching 164

6 Evolutionary Origins of the HO Gene and

Other Components of the MAT Switching System 166

7 Linkage of an HM Cassette with the

Recombination Enhancer (RE) 167

8 The HO Endonuclease Site in MAT a 1 168

9 Relationship of HO to VDE 169

10 Hypothesis for the Evolutionary Origin of HO 172

References 173

Engineering Homing Endonucleases for Genomic Applications 177

Frederick S. Gimble

1 Introduction 177

2 The Modular Organization of Homing

Endonucleases and Their Endonucleolytic Activity 178

3 Probing the Modular Structure of Homing

Endonucleases by Protein Engineering 180

4 Engineering Homing Enzymes with Novel Functions. ... 182

4.1 Changing the Recognition Site Specificity of Homing Endonucleases 182

4.1.1 Altering Homing Endonuclease Specificity by Domain Shuffling 182

4.1.2 Altering Homing Endonuclease Specificity

Using Genetic Screens and Selections 185

4.2 Introducing Molecular Switches into

Homing Endonucleases 189

5 Conclusions and Future Prospects 190

References 190

Inteins - A Historical Perspective 193

Francine B. Perler

1 Introduction 194

2 Efforts To Prove That Splicing Is Post-translational 194

3 Intein Motifs and Conserved Residues 195

4 Intein Polymorphisms and Non-canonical Inteins 197

5 Criteria for Intein Designation 198

6 The Minimal Splicing Element 198

7 Splicing of Split Inteins: Trans-Splicing 199

8 The Influence of Exteins and

Insertion Site Characteristics 200

9 The Challenge of Deciphering the Protein-Splicing Mechanism 201

10 Splicing of Non-canonical Inteins 204

11 Why Are Inteins So Robust? 205

12 Control, Control, Control 205

13 Perspectives for the Future 206

References 207

Origin and Evolution of Inteins and Other Hint Domains 211

Baraket Dassa, Shmuel Pietrokovski

1 Introduction 211

2 Hint Domain Families 213

2.1 Inteins 213

2.1.1 Inteins Include Different Domains 213

2.1.2 Intein Protein Hosts and Insertion Points 214

2.1.3 Inteins Are Sporadically Distributed 215

2.1.4 A Non-selfish Intein-Derived Protein 216

2.1.5 Split Inteins 217

2.2 Hog-Hint Domains 218

2.2.1 Phylogenetic Distribution of Hog-Hint Domains 218

2.3 BIL-Hint Domains 220

2.3.1 Phylogenetic Distribution of BIL Domains 221

2.3.2 Protein Distribution of BIL Domains 222

2.3.3 Biochemical Activity and Biological Roles of BIL Domains 222

2.4 Other Hint Domains 224

3 Origin of the Hint Domains 225

3.1 Features of the Progenitor Hint Domain 225

3.2 Emergence of the Progenitor Hint Domain 227

References 229

Biochemical Mechanisms of Intein-Mediated Protein Splicing 233

Kenneth V. Mills, Henry Paulus

1 Conserved Features of Intein Structure 234

2 The Canonical Protein-Splicing Mechanism 234

2.1 First Step of Protein Splicing-N/O or N/SAcyl Shift.. .. 235

2.2 Second Step of Protein Splicing - Transesterification 238

2.3 Third Step of Protein Splicing - Asparagine Cyclization . . 240

2.4 Finishing Reaction 241

2.5 Association of Split Inteins 242

3 Non-canonical Inteins and Their Mechanisms 243

3.1 Substitution of the N-Terminal Nucleophile 243

3.2 Substitution of the C-Terminal Asparagine 245

3.3 Hedgehog Autoprocessing Domains 245

3.4 Bacterial Intein-Like Domains 246

4 Protein Splicing as a System 247

4.1 Side Reactions 247

4.2 What Coordinates the Steps in the

Protein-Splicing Pathway? 249

5 Conclusions 251

References 252

The Structure and Function of Intein-Associated

Homing Endonucleases 25 7

Carmen M. Moure, Florante A. Quiocho

1 Introduction 257

2 Architecture of Inteins: A Two-Domain Organization. . . . 258

2.1 The Splicing Domain 260

2.2 Endonuclease Domain 261

3 DNA Binding to the Splicing and

Endonuclease Domains of PI-SceI 261

3.1 Protein-DNA Contacts Across the

Splicing and Endonuclease Domains 263

3.2 Protein Conformational Changes 263

3.3 DNA Bending 264

3.4 DNA Recognition 266

4 DNA Cleavage 266

4.1 Active Sites 266

4.2 Divalent Metal Binding at the Active Sites 267

References 269

Harnessing Inteins for Protein Purification and Characterization. . . 273

Shaorong Chong, Ming-Qun Xu

1 Introduction 273

2 Intein Fusion Systems for Protein Purification 273

2.1 C-Terminal Fusion System 275

2.2 N-Terminal Fusion System 277

2.3 Choosing an Appropriate Intein Fusion System 278

2.4 Choosing an Appropriate Residue at the Fusion Junction . 280

2.5 Conditions for Intein Cleavage 281

2.6 Intein Fusion Systems for

High-Throughput and Large-Scale Applications 283

3 Protein trans-Splicing and Cleavage Systems 283

4 Intein-Mediated Protein Ligation (IPL) 284

4.1 Intein-Mediated Peptide Array (IPA) 286

4.2 Kinase Assays Using Carrier Protein-Peptide Substrates . . 288

4.3 Purification of Peptide-Specific Antibody 289

5 Remarks and Conclusions 289

References 290

Production of Cyclic Proteins and Peptides 293

Ali Tavassoli, Todd A. Naumann, Stephen J. Benkovic

1 Introduction 293

1.1 Naturally Occurring Cyclic Peptides 293

1.2 Intein-Mediated Cyclization 295

2 Intein-Mediated Ligation 295

3 TWIN 297

4 trans- and czs-Splicing 298

5 Cyclization with Artificially Split Inteins 299

6 SICLOPPS 300

7 Summary 302

References 302

Inteins for Split-Protein Reconstitutions and Their Applications . . . 307

Takeaki Ozawa,Yoshio Umezawa

1 Introduction 307

2 General Characteristics of Protein Splicing 308

3 Reporter Protein Reconstitution 309

3.1 Detection of Protein-Protein Interactions 311

3.2 Protein Splicing in Intracellular Organelles 315

3.2.1 Identification of Organelle-Localized

Proteins from cDNA Libraries 315

3.2.2 Detection of Protein Nuclear Transport 317

3.2.3 frans-Splicing in the Chloroplast in Plant Cells 319

3.3 Screening of Potential Antimycobacterial Agents 320

4 Future Directions 321

References 322

Intein Reporter and Selection Systems 325

David W. Wood, Georgios Skretas

1 Introduction 325

2 Systems for Direct Observation of Intein Function 327

3 Selection and Reporter Systems for Intein Function 328

3.1 Insertion into the LacZa Gene 331

3.2 Insertion into the Saccharomyces cerevisiae VAT2 Gene .. 331

3.3 Thymidylate Synthase System for Selection in vivo 332

3.4 Kanamycin Resistance: the ORFTRAP System 333

3.5 DNA gyraseA: Negative Selection for Splicing in vivo. . . . 334

3.6 Bacterial ccdB: Negative Selection for Splicing or Cleaving 335

3.7 T4 DNA Polymerase:

Negative-Splicing Selection in vivo 336

3.8 Yeast Gal4 Gene:

Temperature-Sensitive Splicing in vivo 337

3.9 Green Fluorescent Protein:

Reporter for Splicing in vitro 338

3.10 frans-Splicing Intein Systems 339

4 Summary 340

References 341

Industrial Applications of Intein Technology 345

David W. Wood, Sarah W. Harcum, Georges Belfort

1 Introduction 346

2 Scale-Up of Intein Processes 346

2.1 Conventional Affinity Tag Processes 347

2.2 Intein-Mediated Protein Purification 347

2.2.1 Modeling Large-Scale Intein Bioseparations 348

2.2.2 Economics of the IMPACT Process Scale-Up 350

2.3 Economic Optimization of Intein-Based Bioseparations . . 350

2.3.1 Buffers 351

2.3.2 Resins 353

2.3.3 Alternate Intein-Cleaving Modes 354

2.4 Economics of an Optimal Large-Scale Intein Process 355

2.5 Additional Considerations for Intein Process Scale-Up . . . 356

3 Scale-Down of Intein Processes 357

3.1 Microfluidics 358

3.2 Protein Micro-arrays 361

4 Summary 362

References 363

Subject Index

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