Essent ia l GENES

Benjamin Lewin

Upper Saddle River, NJ 07458

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© 2006 by Benjamin Lewin Published by Pearson Education, Inc.Pearson Prentice Hall Pearson Education, Inc.Upper Saddle River, NJ 07458

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Brief ContentsPart 1 Genes

1 DNA Is the Hereditary Material 1

2 Genes Code for Proteins 233 Genes May Be Interrupted 384 The Content of the Genome 545 Genome Sequences

and Gene Numbers 726 Clusters and Repeats 90

Part 2 Proteins

7 Messenger RNA 1138 Protein Synthesis 1349 Using the Genetic Code 16210 Protein Localization

Requires Special Signals 181

Part 3 Gene Expression

11 Transcription 19412 The Operon 22213 Regulatory RNA 24314 Phage Strategies 258

Part 4 DNA Replication andRecombination

15 The Replicon 28016 Extrachromosomal

Replicons 29017 Bacterial Replication Is

Connected to the Cell Cycle 30318 DNA Replication 317

19 Homologous and Site-Specific Recombination 334

20 Repair Systems Handle Damage to DNA 356

21 Transposons 37222 Retroviruses and

Retroposons 39223 Recombination in

the Immune System 409

Part 5 Eukaryotic GeneExpression

24 Promoters and Enhancers 42925 Regulating Eukaryotic

Transcription 44926 RNA Splicing and

Processing 46827 Catalytic RNA 490

Part 6 The Nucleus

28 Chromosomes 50729 Nucleosomes 52630 Chromatin Structure Is

a Focus for Regulation 55031 Epigenetic Effects Are

Inherited 56532 Genetic Engineering 581

Glossary G-1

Credits C-1

Index I-1

v

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vii

Contents

Part 1 Genes

3

2

1DNA Is the Hereditary Material

1.1 Introduction 11.2 DNA Is the Genetic Material of Bacteria 21.3 DNA Is the Genetic Material of Viruses 31.4 DNA Is the Genetic Material of Animal Cells 41.5 Polynucleotide Chains Have Nitrogenous Bases Linked to

a Sugar-Phosphate Backbone 41.6 DNA Is a Double Helix 51.7 Supercoiling Affects the Structure of DNA 81.8 The Structure of DNA Allows Replication and Transcription 91.9 DNA Replication Is Semiconservative 111.10 DNA Strands Separate at the Replication Fork 121.11 Genetic Information Can Be Provided by DNA or RNA 121.12 Nucleic Acids Hybridize by Base Pairing 141.13 Mutations Change the Sequence of DNA 151.14 Mutations May Affect Single Base Pairs or Longer Sequences 161.15 The Effects of Mutations Can be Reversed 181.16 Mutations Are Concentrated at Hotspots 191.17 Many Hotspots Result from Modified Bases 201.18 Genomes Vary Greatly in Size 201.19 Summary 22

Genes Code for Proteins2.1 Introduction 232.2 A Gene Codes for a Single Polypeptide 242.3 Mutations in the Same Gene Cannot Complement 252.4 Mutations May Cause Loss-of-Function or Gain-of-Function 262.5 A Locus May Have Many Different Mutant Alleles 272.6 A Locus May Have More Than One Wild-Type Allele 282.7 Recombination Occurs by Physical Exchange of DNA 292.8 The Probability of Recombination Depends on Distance Apart 302.9 The Genetic Code Is Triplet 312.10 Every Sequence Has Three Possible Reading Frames 332.11 Several Processes Are Required to Express the Protein Product of a Gene 342.12 Proteins Are trans-Acting but Sites on DNA Are cis-Acting 352.13 Summary 37

Genes May Be Interrupted3.1 Introduction 383.2 Interrupted Genes Were First Detected by Comparing mRNA and DNA 393.3 Interrupted Genes Are Much Longer Than the Corresponding mRNAs 413.4 Organization of Interrupted Genes Is Often Conserved 433.5 Exon Sequences Are Conserved but Introns Vary 443.6 Genes Show a Wide Distribution of Lengths 45

Preface xvii

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3.7 Some DNA Sequences Code for More Than One Protein 463.8 How Did Interrupted Genes Evolve? 483.9 Some Exons Can Be Equated with Protein Functions 503.10 The Members of a Gene Family Have a Common Organization 513.11 Pseudogenes Are Dead Ends of Evolution 523.12 Summary 53

6

5

4The Content of the Genome

4.1 Introduction 544.2 Genomes Can Be Mapped by Linkage, Restriction Cleavage, or DNA Sequence 554.3 Individual Genomes Show Extensive Variation 564.4 RFLPs and SNPs Can Be Used for Genetic Mapping 584.5 Why Are Genomes So Large? 604.6 Eukaryotic Genomes Contain Both Nonrepetitive and Repetitive DNA Sequences 614.7 Genes Can Be Isolated by the Conservation of Exons 624.8 Genes Involved in Diseases Can Be Identified by Comparing Patient

DNA with Normal DNA 644.9 The Conservation of Genome Organization Helps to Identify Genes 654.10 Organelles Have DNA 674.11 Mitochondrial Genomes Are Circular DNAs That Code for Organelle Proteins 684.12 The Chloroplast Genome Codes for Many Proteins and RNAs 704.13 Organelles Evolved by Endosymbiosis 704.14 Summary 71

Genome Sequences and Gene Numbers5.1 Introduction 725.2 Bacterial Gene Numbers Range Over an Order of Magnitude 735.3 Total Gene Number Is Known for Several Eukaryotes 755.4 The Human Genome Has Fewer Genes Than Expected 765.5 How Are Genes and Other Sequences Distributed in the Genome? 785.6 The Y Chromosome Has Several Male-Specific Genes 795.7 How Many Different Types of Genes Are There? 815.8 More Complex Species Evolve by Adding New Gene Functions 835.9 How Many Genes Are Essential? 845.10 About 10,000 Genes Are Expressed at Widely Different Levels

in a Eukaryotic Tissue 865.11 Expressed Gene Number Can Be Measured en masse 885.12 Summary 89

Clusters and Repeats6.1 Introduction 906.2 Gene Duplication Is a Major Force in Evolution 926.3 Globin Clusters Are Formed by Duplication and Divergence 926.4 Sequence Divergence Is the Basis for the Evolutionary Clock 946.5 The Rate of Neutral Substitution Can Be Measured from Divergence

of Repeated Sequences 976.6 Unequal Crossing-Over Rearranges Gene Clusters 986.7 Genes for rRNA Form Tandem Repeats Including an Invariant Transcription Unit 1006.8 Crossover Fixation Could Maintain Identical Repeats 1026.9 Satellite DNAs Often Lie in Heterochromatin 1046.10 Arthropod Satellites Have Very Short Identical Repeats 1066.11 Mammalian Satellites Consist of Hierarchical Repeats 1076.12 Minisatellites Are Useful for Genetic Mapping 1096.13 Summary 112

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Part 2 Proteins

7Messenger RNA

7.1 Introduction 1137.2 mRNA Is Produced by Transcription and Is Translated 1147.3 Transfer RNA Forms a Cloverleaf 1157.4 The Acceptor Stem and Anticodon Are at Ends of the Tertiary Structure 1177.5 Messenger RNA Is Translated by Ribosomes 1187.6 Many Ribosomes Bind to One mRNA 1197.7 The Life Cycle of Bacterial Messenger RNA 1217.8 Eukaryotic mRNA Is Modified During or After Its Transcription 1237.9 The 5' End of Eukaryotic mRNA Is Capped 1247.10 The Eukaryotic mRNA 3' Terminus Is Polyadenylated 1257.11 Bacterial mRNA Degradation Involves Multiple Enzymes 1267.12 Two Pathways Degrade Eukaryotic mRNA 1277.13 Nonsense Mutations Trigger a Eukaryotic Surveillance System 1297.14 Eukaryotic RNAs Are Transported 1307.15 mRNAs Can Be Localized Within a Cell 1317.16 Summary 133

9

8Protein Synthesis

8.1 Introduction 1348.2 Protein Synthesis Occurs by Initiation, Elongation, and Termination 1368.3 Special Mechanisms Control the Accuracy of Protein Synthesis 1388.4 Initiation in Bacteria Needs 30S Subunits and Accessory Factors 1398.5 A Special Initiator tRNA Starts the Polypeptide Chain 1418.6 mRNA Binds a 30S Subunit to Create the Binding Site

for a Complex of IF-2 and fMet-tRNAf 1428.7 Small Eukaryotic Subunits Scan for Initiation Sites on mRNA 1448.8 Elongation Factor Tu Loads Aminoacyl-tRNA into the A Site 1468.9 The Polypeptide Chain Is Transferred to Aminoacyl-tRNA 1478.10 Translocation Moves the Ribosome 1488.11 Elongation Factors Bind Alternately to the Ribosome 1508.12 Uncharged tRNA Causes the Ribosome to Trigger the Stringent Response 1508.13 Three Codons Terminate Protein Synthesis and Are Recognized

by Protein Factors 1538.14 Ribosomal RNA Pervades Both Ribosomal Subunits 1558.15 Ribosomes Have Several Active Centers 1568.16 Both rRNAs Play Active Roles in Protein Synthesis 1588.17 Summary 160

Using the Genetic Code9.1 Introduction 1629.2 Related Codons Represent Related Amino Acids 1639.3 Codon–Anticodon Recognition Involves Wobbling 1649.4 tRNA Contains Modified Bases 1659.5 Modified Bases Affect Anticodon–Codon Pairing 1679.6 There Are Sporadic Alterations of the Universal Code 1689.7 Novel Amino Acids Can Be Inserted at Certain Stop Codons 1699.8 tRNAs Are Charged with Amino Acids by Synthetases 1709.9 Aminoacyl-tRNA Synthetases Fall into Two Groups 1719.10 Synthetases Use Proofreading to Improve Accuracy 1729.11 Suppressor tRNAs Have Mutated Anticodons That Read New Codons 174

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9.12 Recoding Changes Codon Meanings 1769.13 Frameshifting Occurs at Slippery Sequences 1779.14 Bypassing Involves Ribosome Movement 1799.15 Summary 180

10Protein Localization Requires Special Signals

10.1 Introduction 18110.2 Protein Translocation May Be Post-translational or Co-translational 18210.3 The Signal Sequence Interacts with the SRP 18410.4 The SRP Interacts with the SRP Receptor 18610.5 The Translocon Forms a Pore 18810.6 Post-translational Membrane Insertion Depends on Leader Sequences 18910.7 Bacteria Use Both Co-translational and Post-translational Translocation 19110.8 Summary 193

Part 3 Gene Expression

12

11Transcription

11.1 Introduction 19411.2 Transcription Occurs by Base Pairing in a “Bubble” of Unpaired DNA 19611.3 The Transcription Reaction Has Three Stages 19611.4 A Model for Enzyme Movement Is Suggested by the Crystal Structure 19811.5 RNA Polymerase Consists of the Core Enzyme and Sigma Factor 20011.6 How Does RNA Polymerase Find Promoter Sequences? 20211.7 Sigma Factor Controls Binding to DNA 20311.8 Promoter Recognition Depends on Consensus Sequences 20511.9 Promoter Efficiencies Can Be Increased or Decreased by Mutation 20711.10 Supercoiling Is an Important Feature of Transcription 20811.11 Substitution of Sigma Factors May Control Initiation 20911.12 Sigma Factors Directly Contact DNA 21211.13 There Are Two Types of Terminators in E. coli 21311.14 Intrinsic Termination Requires a Hairpin and U-Rich Region 21411.15 How Does Rho Factor Work? 21511.16 Antitermination Is a Regulatory Event 21611.17 Summary 220

The Operon12.1 Introduction 22212.2 Structural Gene Clusters Are Coordinately Controlled 22412.3 The lac Genes Are Controlled by a Repressor 22512.4 The lac Operon Can Be Induced 22612.5 Repressor Is Controlled by a Small Molecule Inducer 22712.6 cis-Acting Constitutive Mutations Identify the Operator 22812.7 trans-Acting Mutations Identify the Regulator Gene 22912.8 Repressor Is a Tetramer Made of Two Dimers 23112.9 Repressor Binding to the Operator Is Regulated

by an Allosteric Change in Conformation 23212.10 Repressor Binds to Three Operators and Interacts with RNA Polymerase 23412.11 The Operator Competes with Low-Affinity Sites to Bind Repressor 23512.12 Repression Can Occur at Multiple Loci 23712.13 Operons May Be Repressed or Induced 23812.14 Cyclic AMP Is an Inducer That Activates CRP to Act at Many Operons 23912.15 Translation Can Be Regulated 24012.16 Summary 241

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16

15

14

13Regulatory RNA

13.1 Introduction 24313.2 Alternative Secondary Structures Can Affect Translation or Transcription 24413.3 Termination of B. Subtilis Trp Genes Is Controlled by Tryptophan and by tRNATrp 24513.4 The E. Coli tryptophan Operon Is Controlled by Attenuation 24613.5 Attenuation Can Be Controlled by Translation 24713.6 Antisense RNA Can Be Used to Inactivate Gene Expression 25013.7 Small RNA Molecules Can Regulate Translation 25113.8 Bacteria Contain Regulator RNAs 25213.9 MicroRNAs Are Regulators in Many Eukaryotes 25413.10 RNA Interference Is Related to Gene Silencing 25513.11 Summary 257

Phage Strategies14.1 Introduction 25814.2 Lytic Development Is Divided into Two Periods 25914.3 Lytic Development Is Controlled by a Cascade 26114.4 Two Types of Regulatory Event Control the Lytic Cascade 26214.5 Lambda Uses Immediate Early and Delayed Early Genes

for Both Lysogeny and the Lytic Cycle 26414.6 The Lytic Cycle Depends on Antitermination 26414.7 Lysogeny Is Maintained by Repressor Protein 26614.8 The Repressor and Its Operators Define the Immunity Region 26714.9 The DNA-Binding Form of Repressor Is a Dimer 26814.10 Repressor Uses a Helix-Turn-Helix Motif to Bind DNA 26914.11 Repressor Dimers Bind Cooperatively to the Operator 27014.12 Repressor Maintains an Autogenous Circuit 27214.13 Cooperative Interactions Increase the Sensitivity of Regulation 27214.14 The cII and cIII Genes Are Needed to Establish Lysogeny 27314.15 Lysogeny Requires Several Events 27414.16 The Cro Repressor Is Needed for Lytic Infection 27614.17 What Determines the Balance between Lysogeny and the Lytic Cycle? 27714.18 Summary 278

Part 4 DNA Replication and RecombinationThe Replicon

15.1 Introduction 28015.2 An Origin Usually Initiates Bidirectional Replication 28115.3 The Bacterial Genome Is a Single Circular Replicon 28215.4 Methylation of the Bacterial Origin Regulates Initiation 28415.5 Each Eukaryotic Chromosome Contains Many Replicons 28515.6 Replication Origins Bind the ORC 28615.7 Licensing Factor Controls Rereplication and Consists of MCM Proteins 28715.8 Summary 289

Extrachromosomal Replicons16.1 Introduction 29016.2 The Ends of Linear DNA Are a Problem for Replication 29116.3 Terminal Proteins Enable Initiation at the Ends of Viral DNAs 29216.4 Rolling Circles Produce Multimers of a Replicon 29316.5 Rolling Circles Are Used to Replicate Phage Genomes 29516.6 The F Plasmid Is Transferred by Conjugation between Bacteria 296

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16.7 Conjugation Transfers Single-Stranded DNA 29716.8 The Ti Bacterial Plasmid Transfers Genes into Plant Cells 29816.9 Transfer of T-DNA Resembles Bacterial Conjugation 29916.10 Summary 302

19

18

17Bacterial Replication Is Connected to the Cell Cycle

17.1 Introduction 30317.2 Bacteria Can Have Multiforked Chromosomes 30417.3 The Septum Divides a Bacterium into Progeny

Each Containing a Chromosome 30517.4 Mutations in Division or Segregation Affect Cell Shape 30617.5 FtsZ Is Necessary for Septum Formation 30717.6 min Genes Regulate the Location of the Septum 30817.7 Chromosomal Segregation May Require Site-Specific Recombination 30917.8 Partitioning Separates the Chromosomes 31017.9 Single-Copy Plasmids Have a Partitioning System 31217.10 Plasmid Incompatibility Is Determined by the Replicon 31417.11 How Do Mitochondria Replicate and Segregate? 31517.12 Summary 316

DNA Replication18.1 Introduction 31718.2 DNA Polymerases Are the Enzymes That Make DNA 31818.3 DNA Polymerases Control the Fidelity of Replication 31918.4 DNA Polymerases Have a Common Structure 32018.5 The Two New DNA Strands Have Different Modes of Synthesis 32118.6 Replication Requires a Helicase and Single-Strand Binding Protein 32218.7 Priming Is Required to Start DNA Synthesis 32318.8 DNA Polymerase Holoenzyme Consists of Subcomplexes 32418.9 The Clamp Controls Association of Core Enzyme with DNA 32518.10 Coordinating the Synthesis of the Lagging and Leading Strands 32618.11 Okazaki Fragments Are Linked by Ligase 32818.12 Separate Eukaryotic DNA Polymerases Undertake Initiation and Elongation 32918.13 Creating the Replication Forks at an Origin 33018.14 The Primosome Is Needed to Restart Replication 33118.15 Summary 333

Homologous and Site-Specific Recombination19.1 Introduction 33419.2 Breakage and Reunion Involves Heteroduplex DNA 33519.3 Double-Strand Breaks Initiate Recombination 33819.4 Recombining Chromosomes Are Connected by the Synaptonemal Complex 33919.5 The Synaptonemal Complex Forms after Double-Strand Breaks 34019.6 RecBCD Generates Free Ends for Recombination 34219.7 Strand-Transfer Proteins Catalyze Single-Strand Assimilation 34319.8 The Ruv System Resolves Holliday Junctions 34419.9 Topoisomerases Relax or Introduce Supercoils in DNA 34519.10 Topoisomerases Break and Reseal Strands 34619.11 Site-Specific Recombination Resembles Topoisomerase Activity 34719.12 Specialized Recombination in Phage Lambda Involves Specific Sites 34919.13 Yeast Mating Type Is Changed by Recombination 35119.14 Unidirectional Transposition Is Initiated by the Recipient MAT Locus 35319.15 Summary 354

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Repair Systems Handle Damage to DNA20.1 Introduction 35620.2 Mutational Damage Falls into Two General Types 35820.3 Excision Repair Systems in E. coli 36020.4 Base Flipping Is Used by Methylases and Glycosylases 36120.5 Error-Prone Repair and Mutator Phenotypes 36220.6 Controlling the Direction of Mismatch Repair 36320.7 Recombination-Repair Systems in E. coli 36520.8 Recombination Is Important for Correcting Replication Errors 36620.9 Eukaryotic Cells Have Conserved Repair Systems 36720.10 A Common System Repairs Double-Strand Breaks 36820.11 Defects in Repair Systems Cause Mutations to Accumulate in Tumors 37020.12 Summary 371

23

22

21

20

Transposons21.1 Introduction 37221.2 Insertion Sequences Are Simple Transposition Modules 37321.3 Composite Transposons Have IS Modules 37421.4 Transposition Occurs by Both Replicative and Nonreplicative Mechanisms 37521.5 Transposons Cause Rearrangement of DNA 37721.6 Common Intermediates for Transposition 37821.7 Replicative Transposition Proceeds Through a Cointegrate 38021.8 Nonreplicative Transposition Proceeds by Breakage and Reunion 38121.9 TnA Transposition Requires Transposase and Resolvase 38221.10 Controlling Elements in Maize Cause Breakage and Rearrangements 38421.11 Controlling Elements Form Families of Transposons 38621.12 Transposition of P Elements Causes Hybrid Dysgenesis 38821.13 Summary 391

Retroviruses and Retroposons22.1 Introduction 39222.2 The Retrovirus Life Cycle Involves Transposition-Like Events 39322.3 Retroviral Genes Code for Polyproteins 39422.4 Viral DNA Is Generated by Reverse Transcription 39522.5 Viral DNA Integrates Into the Chromosome 39822.6 Retroviruses May Transduce Cellular Sequences 39922.7 Yeast Ty Elements Resemble Retroviruses 40022.8 Many Transposable Elements Reside in D. melanogaster 40122.9 Retroposons Fall into Three Classes 40222.10 The Alu Family Has Many Widely Dispersed Members 40422.11 Processed Pseudogenes Originated as Substrates for Transposition 40522.12 LINES Use an Endonuclease to Generate a Priming End 40622.13 Summary 408

Recombination in the Immune System23.1 Introduction 40923.2 Immunoglobulin Genes Are Assembled from Their Parts in Lymphocytes 41123.3 Light Chains Are Assembled by a Single Recombination 41323.4 Heavy Chains Are Assembled by Two Recombinations 41523.5 Recombination Generates Extensive Diversity 41623.6 Immune Recombination Uses Two Types of Consensus Sequence 41723.7 Recombination Generates Deletions or Inversions 41823.8 The RAG Proteins Catalyze Breakage and Reunion 419

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23.9 Class Switching Is Caused by a Novel Type of DNA Recombination 42223.10 Somatic Mutation Is Induced by Cytidine Deaminase and Uracil Glycosylase 42423.11 Avian Immunoglobulins Are Assembled from Pseudogenes 42523.12 T-Cell Receptors Are Related to Immunoglobulins 42723.13 Summary 428

Part 5 Eukaryotic Gene Expression

26

25

24Promoters and Enhancers

24.1 Introduction 42924.2 Eukaryotic RNA Polymerases Consist of Many Subunits 43124.3 RNA Polymerase I Has a Bipartite Promoter 43224.4 RNA Polymerase III Uses Both Downstream and Upstream Promoters 43324.5 The Startpoint for RNA Polymerase II 43524.6 TBP Is a Component of TFIID and Binds the TATA Box 43624.7 The Basal Apparatus Assembles at the Promoter 43824.8 Initiation Is Followed by Promoter Clearance 43924.9 Short Sequence Elements Bind Activators 44024.10 Enhancers Contain Bidirectional Elements That Assist Initiation 44224.11 Enhancers Contain the Same Elements That Are Found at Promoters 44324.12 Enhancers Work by Increasing the Concentration

of Activators Near the Promoter 44524.13 CpG Islands Are Regulatory Targets 44624.14 Summary 448

Regulating Eukaryotic Transcription25.1 Introduction 44925.2 There Are Several Types of Transcription Factors 45025.3 Independent Domains Bind DNA and Activate Transcription 45125.4 Activators Interact with the Basal Apparatus 45225.5 Response Elements Are Recognized by Activators 45425.6 There Are Many Types of DNA-Binding Domains 45625.7 A Zinc Finger Motif Is a DNA-Binding Domain 45825.8 Some Steroid Hormone Receptors Are Transcription Factors 45925.9 Zinc Fingers of Steroid Receptors Use a Combinatorial Code 46025.10 Binding to the Response Element Is Activated by Ligand Binding 46225.11 Homeodomains Bind Related Targets in DNA 46225.12 Helix-Loop-Helix Proteins Interact by Combinatorial Association 46425.13 Leucine Zippers Are Involved in Dimer Formation 46525.14 Summary 466

RNA Splicing and Processing26.1 Introduction 46826.2 Nuclear Splice Junctions Are Short Sequences 46926.3 Splice Junctions Are Read in Pairs 47026.4 pre-mRNA Splicing Proceeds Through a Lariat 47126.5 snRNAs Are Required for Splicing 47326.6 U1 snRNP Initiates Splicing 47426.7 The E Complex Commits an RNA to Splicing 47526.8 5 snRNPs Form the Spliceosome 47626.9 Splicing Is Connected to Export of mRNA 47826.10 Group II Introns Autosplice via Lariat Formation 47926.11 Alternative Splicing Involves Differential Use of Splice Junctions 481

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26.12 Trans-Splicing Reactions Use Small RNAs 48226.13 Yeast tRNA Splicing Involves Cutting and Rejoining 48326.14 The 3' Ends of mRNAs Are Generated by Cleavage and Polyadenylation 48626.15 Small RNAs Are Required for rRNA Processing 48726.16 Summary 489

29

28

27Catalytic RNA

27.1 Introduction 49027.2 Group I Introns Undertake Self-Splicing by Transesterification 49127.3 Group I Introns Form a Characteristic Secondary Structure 49327.4 Ribozymes Have Various Catalytic Activities 49427.5 Some Group I Introns Code for Endonucleases That Sponsor Mobility 49627.6 Some Group II Introns Code for Reverse Transcriptases 49827.7 Some Autosplicing Introns Require Maturases 49927.8 Viroids Have Catalytic Activity 49927.9 RNA Editing Occurs at Individual Bases 50127.10 RNA Editing Can Be Directed by Guide RNAs 50227.11 Protein Splicing Is Autocatalytic 50527.12 Summary 506

Part 6 The NucleusChromosomes

28.1 Introduction 50728.2 Viral Genomes Are Packaged into Their Coats 50828.3 The Bacterial Genome Is a Supercoiled Nucleoid 51028.4 Eukaryotic DNA Has Loops and Domains Attached to a Scaffold 51228.5 Chromatin Is Divided into Euchromatin and Heterochromatin 51328.6 Chromosomes Have Banding Patterns 51528.7 Lampbrush Chromosomes Are Extended 51628.8 Polytene Chromosomes Form Bands That Puff at Sites of Gene Expression 51728.9 Centromeres Often Have Extensive Repetitive DNA 51928.10 S. cerevisiae Centromeres Have Short Protein-Binding DNA Sequences 52028.11 Telomeres Have Simple Repeating Sequences 52228.12 Telomeres Are Synthesized by a Ribonucleoprotein Enzyme 52328.13 Summary 525

Nucleosomes29.1 Introduction 52629.2 The Nucleosome Is the Subunit of all Chromatin 52729.3 DNA Is Coiled in Arrays of Nucleosomes 52829.4 Nucleosomes Have a Common Structure 52929.5 DNA Structure Varies on the Nucleosomal Surface 53029.6 The Nucleosome Absorbs Some Supercoiling 53229.7 Organization of the Core Particle 53329.8 The Path of Nucleosomes in the Chromatin Fiber 53429.9 Reproduction of Chromatin Requires Assembly of Nucleosomes 53529.10 Do Nucleosomes Lie at Specific Positions? 53829.11 Histone Octamers Are Displaced by Transcription 54029.12 DNAase Hypersensitive Sites Change Chromatin Structure 54229.13 Domains Define Regions That Contain Active Genes 54329.14 Insulators Block the Actions of Enhancers and Heterochromatin 54529.15 An LCR May Control a Domain 54729.16 What Constitutes a Regulatory Domain? 54829.17 Summary 549

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Chromatin Structure Is a Focus for Regulation30.1 Introduction 55030.2 Chromatin Remodeling Is an Active Process 55130.3 There Are Several Chromatin Remodeling Complexes 55230.4 Nucleosome Organization May Be Changed at the Promoter 55430.5 Histone Modification Is a Key Event 55530.6 Histone Acetylation Occurs in Two Circumstances 55530.7 Acetylases Are Associated with Activators 55630.8 Deacetylases Are Associated with Repressors 55830.9 Methylation of Histones and DNA Is Connected 55830.10 Promoter Activation Is an Ordered Series of Events 55930.11 Histone Phosphorylation Affects Chromatin Structure 56030.12 Some Common Motifs Are Found in Proteins That Modify Chromatin 56130.13 Heterochromatin Depends on Interactions with Histones 56230.14 Summary 564

31

30

Epigenetic Effects Are Inherited31.1 Introduction 56531.2 Heterochromatin Propagates from a Nucleation Event 56631.3 Polycomb and Trithorax Are Antagonistic Repressors and Activators 56831.4 X Chromosomes Undergo Global Changes 57031.5 DNA Methylation Is Perpetuated by a Maintenance Methylase 57231.6 DNA Methylation Is Responsible for Imprinting 57431.7 Yeast Prions Show Unusual Inheritance 57631.8 Prions Cause Diseases in Mammals 57831.9 Summary 580

32Genetic Engineering

32.1 Introduction 58132.2 Cloning Vectors Are Used to Amplify Donor DNA 58232.3 Cloning Vectors Can Be Specialized for Different Purposes 58532.4 Transfection Introduces Exogenous DNA into Cells 58732.5 Genes Can Be Injected into Animal Eggs 58932.6 ES Cells Can Be Incorporated into Embryonic Mice 59132.7 Gene Targeting Allows Genes to Be Replaced or Knocked Out 59232.8 Summary 594

Glossary G-1

Credits C-1

Index I-1

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1.1 Introduction

Key Terms

• The genome is the complete set of sequences in the geneticmaterial of an organism. It includes the sequence of each chro-mosome plus any DNA in organelles.

• Nucleic acids are molecules that encode genetic information.They consist of a series of nitrogenous bases connected to ribosemolecules that are linked by phosphodiester bonds. DNA is de-oxyribonucleic acid, and RNA is ribonucleic acid.

• A chromosome is a discrete unit of the genome carrying manygenes. Each chromosome consists of a very long molecule of du-plex DNA and an approximately equal mass of proteins. It is visi-ble as a morphological entity only during cell division.

• A gene (cistron) is the segment of DNA specifying production of apolypeptide chain; it includes regions preceding and following thecoding region (leader and trailer) as well as intervening sequences(introns) between individual coding segments (exons).

The hereditary nature of every living organism is defined by itsgenome, which consists of a long sequence of nucleic acid that providesthe information needed to construct the organism. We use the term “in-formation” because the genome does not itself perform any active rolein building the organism; rather it is the sequence of the individual sub-units (bases) of the nucleic acid that determines hereditary features. Bya complex series of interactions, this sequence is used to produce all theproteins of the organism in the appropriate time and place.

A genome is divided physically into chromosomes and functional-ly into genes. Each chromosome is an independent physical unit that

1DNA Is theHereditaryMaterial

1.1 Introduction1.2 DNA Is the Genetic Material of Bacteria1.3 DNA Is the Genetic Material of Viruses1.4 DNA Is the Genetic Material of Animal

Cells1.5 Polynucleotide Chains Have

Nitrogenous Bases Linked to a Sugar-Phosphate Backbone

1.6 DNA is a double helix1.7 Supercoiling Affects the Structure of

DNA1.8 The Structure of DNA Allows

Replication and Transcription1.9 DNA Replication is Semiconservative1.10 DNA Strands Separate at the

Replication Fork1.11 Genetic Information Can Be Provided

by DNA or RNA1.12 Nucleic Acids Hybridize by Base Pairing1.13 Mutations Change the Sequence of

DNA1.14 Mutations May Affect Single Base

Pairs or Longer Sequences1.15 The Effects of Mutations Can Be

Reversed1.16 Mutations Are Concentrated at

Hotspots1.17 Many Hotspots Result From Modified

Bases1.18 Genomes Vary Greatly in Size1.19 Summary

1

LEWIMC01_001-022hr3 7/2/05 10:57 AM Page 1

A gene is a coding unit

DNA

RNA

Protein

Sequence of nucleotides

Gene Chemical nature

Sequence of nucleotides

Sequence of amino acids

Figure 1.2 A gene codes for an RNA, which may code forprotein.

1995 Bacterial genomes sequenced

2001 Human genome sequenced

1865 Genes are particulate factors

1871 Discovery of nucleic acids

1903 Chromosomes are hereditary units

1910 Genes lie on chromosomes

1913 Chromosomes are linear arrays of genes

1927 Mutations are physical changes in genes

1931 Recombination occurs by crossing over

1945 A gene codes for protein

1951 First protein sequence

1953 DNA is a double helix

1958 DNA replicates semiconservatively

1961 Genetic code is triplet

1977 Eukaryotic genes are interrupted

1944 DNA is the genetic material

1977 DNA can be sequenced

1850

1900

1950

2000

Major events in the genetics century

Figure 1.1 A brief history of genetics.

2 CHAPTER 1 DNA Is the Hereditary Material

carries a DNA sequence that contains many genes. The ultimate de-scription of a genome specifies the DNA sequence of each chromosome.

During the last hundred years, we have progressed from Mendel’sobservation that the gene is a particulate structure, through the discov-ery that it consists of DNA, to Watson and Crick’s model for the doublehelix, and most recently to the determination of the sequence of thehuman genome. Figure 1.1 summarizes the stages in the transition fromthe historical concept of the gene to the modern definition of thegenome.

The first definition of the gene as a functional unit followed fromthe discovery that individual genes are responsible for the productionof specific proteins. The difference in chemical nature between theDNA of the gene and its polypeptide product led to the concept that agene codes for a protein. This in turn led to the discovery of the com-plex apparatus that allows the DNA sequence of a gene to generate theamino acid sequence of a protein.

Understanding the process by which a gene is expressed allows usto define “gene” more rigorously. Figure 1.2 shows the basic theme ofthis book. A gene is a sequence of DNA that produces another nucleicacid, RNA. The DNA has two strands of nucleic acid, and the RNA hasonly one strand. The sequence of the RNA is determined by the se-quence of the DNA (in fact, it is identical to one of the DNA strands).In many—but not all—cases, the RNA is in turn used to direct produc-tion of a protein. So a gene is a sequence of DNA that codes for an RNA;in protein-coding genes, the RNA in turn codes for a protein.

1.2 DNA Is the Genetic Materialof Bacteria

Key Terms• Transformation of bacteria is the acquisition of new genetic ma-

terial by incorporation of added DNA.

• The transforming principle is DNA that is taken up by a bacteri-um and whose expression then changes the properties of therecipient cell.

Key Concepts• Bacterial transformation provided the first proof that DNA is the

genetic material by showing that DNA extracted from one bacter-ial strain can change the genetic properties of a second bacterialstrain.

The idea that genetic material is nucleic acid had its roots in the discov-ery of transformation in 1928. The bacterium Pneumococcus kills miceby causing pneumonia.The virulence of the bacterium is determined byits capsular polysaccharide. This is a component of the surface that al-lows the bacterium to escape destruction by the host. Several types (I,II, III) of Pneumococcus have different capsular polysaccharides. Theyhave a smooth (S) appearance.

Each of the smooth Pneumococcal types can give rise to variantsthat fail to produce the capsular polysaccharide. These bacteria have arough (R) surface (consisting of the material that was beneath the cap-sular polysaccharide). They are avirulent. They do not kill the mice, be-cause the absence of the polysaccharide allows the animals to destroythe bacteria.

LEWIMC01_001-022hr3 7/2/05 10:57 AM Page 2

Infect bacteria withlabeled phages 32P label

in DNA

35S labelin protein

Separate phage coatsand infected bacteria

Phage coatscontain 80%of 35S label

Isolate progenyphage particles

Infected bacteriacontain 70% of32P label

Progeny phageshave 30% of 32Plabel and

Transfection introduces new DNA into cells

Colony of TK+ cells

Add TK+ DNA

Cells that lack TK gene cannot producethymidine kinase and die in absence of thymidine

Some cells take up TK gene; descendants of transfected cell pile up into a colony

Figure 1.6 Eukaryotic cells can acquire a new phenotypeas the result of transfection by added DNA.

4 CHAPTER 1 DNA Is the Hereditary Material

1.4 DNA Is the Genetic Materialof Animal Cells

Key Terms• Transfection of eukaryotic cells is the

acquisition of new genetic information byincorporation of added DNA.

Key Concepts• DNA can be used to introduce new genetic features into animal

cells or whole animals.

When DNA is added to populations of single eukaryotic cells growingin culture, the nucleic acid enters the cells, and in some of them resultsin the production of new protein. Figure 1.6 depicts one of the standardsystems, in which addition of a gene for thymidine kinase to mutantcells that do not have the enzyme results in the production of the corre-sponding protein.

Although for historical reasons these experiments are described astransfection when performed with eukaryotic cells, they are a directcounterpart to bacterial transformation. The DNA that is introducedinto the recipient cell becomes part of its genetic material, and is inher-ited in the same way as any other part. Its expression confers a new traitupon the cells (synthesis of thymidine kinase in the example inFigure 1.6). At first, these experiments were successful only with indi-vidual cells adapted to grow in a culture medium. Since then, however,DNA has been introduced into mouse eggs by microinjection; intro-duced DNA may become a stable part of the genetic material of themouse (see 32.4 Genes can be injected into animal eggs).

Such experiments show directly not only that DNA is the geneticmaterial in eukaryotes, but also that it can be transferred between differ-ent species and yet remain functional.

1.5 Polynucleotide Chains HaveNitrogenous Bases Linked toa Sugar-PhosphateBackbone

Key Concepts• A nucleoside consists of a purine or pyrimidine nitrogenous base

linked to position 1 of a pentose sugar.

• Positions on the ribose ring are described with a prime to dis-tinguish them from positions on the base.

• The difference between DNA and RNA is in the group at the po-sition of the sugar. DNA has a deoxyribose sugar RNAhas a ribose sugar

• A nucleotide consists of a nucleoside linked to a phosphate groupon either the or position of the (deoxy)ribose.

• Successive (deoxy)ribose residues of a polynucleotide chain arejoined by a phosphate group between the position of one sugarand the position of the next sugar.

• One end of the chain (conventionally the left) has a free end andthe other end has a free end.

• DNA contains the four bases adenine, guanine, cytosine, andthymine; RNA has uracil instead of thymine.

3¿5¿

5¿3¿

3¿5¿

(2¿ ¬ OH).(2¿ ¬ H);

2¿

(¿)

LEWIMC01_001-022hr3 7/2/05 10:57 AM Page 4

DNA Is a Double Helix SECTION 1.6 5

The basic building block of nucleic acids is the nucleotide.This has three components:

• a nitrogenous base;• a sugar;• and a phosphate.

The nitrogenous base is a purine or pyrimidine ring.The base is linked to position 1 on a pentose sugar by a gly-cosidic bond from of pyrimidines or of purines. Toavoid ambiguity between the numbering systems of theheterocyclic rings and the sugar, positions on the pentoseare given a prime

Nucleic acids are named for the type of sugar; DNAhas –deoxyribose, whereas RNA has ribose. The differ-ence is that the sugar in RNA has an OH group at the position of the pentose ring. The sugar can be linked by its

or position to a phosphate group.A nucleic acid consists of a long chain of nucleotides.

Figure 1.7 shows that the backbone of the polynucleotidechain consists of an alternating series of pentose (sugar) andphosphate residues. This is constructed by linking the po-sition of one pentose ring to the position of the next pen-tose ring via a phosphate group. So the sugar-phosphatebackbone is said to consist of phosphodiester linkages.The nitrogenous bases “stick out” from the backbone.

Each nucleic acid contains four types of base. The sametwo purines, adenine and guanine, are present in both DNAand RNA. The two pyrimidines in DNA are cytosine andthymine; in RNA uracil is found instead of thymine. Theonly difference between uracil and thymine is that thyminehas a methyl group at position and uracil has none. Thebases are usually referred to by their initial letters. DNAcontains A, G, C,T, while RNA contains A, G, C, U.

The terminal nucleotide at one end of the chain has afree group; the terminal nucleotide at the other end hasa free group. It is conventional to write nucleic acid sequences in the

direction—that is, from the terminus at the left to the ter-minus at the right.

1.6 DNA Is a Double HelixKey Terms• Deoxyribonucleic acid (DNA) is a nucleic acid molecule consist-

ing of long chains of polymerized (deoxyribo)nucleotides. Indouble-stranded DNA the two strands are held together by hydro-gen bonds between complementary nucleotide base pairs.

• Base pairing describes the specific (complementary) interactionsof adenine with thymine or of guanine with cytosine in a DNA dou-ble helix (thymine is replaced by uracil in double helical RNA).

• Antiparallel strands of the double helix are organized in oppositeorientation, so that the end of one strand is aligned with the end of the other strand.

• The minor groove of DNA is 12 Å across.• The major groove of DNA is 22 Å across.• A helix is said to be right-handed if the turns run clockwise along

the helical axis.

3¿5¿

3¿5¿5¿ : 3¿3¿

5¿

C5

5¿–3¿

3¿5¿

3¿5¿

2¿2¿

1¿2.

N9N1

5�

3�

CHHC

N

N N

NC

C

C

NH2�O

CH2

NC

O

CHC

N

C

H

NH2

O O

P

O

O

O

P

O

O

CHC

N

N N

NC

C

C

NH2

NH2

CH2

O

O

O

CH2

O O

�O

P O

O

�O

P O�O

A polynucleotide has a repeating structure

Nucleotidesubunit

Pyrimidinebase

Purinebase

Sugar-phosphatebackbone

5�–3� phospho-diester bonds

Figure 1.7 A polynucleotide chain consists of a series of sugar-phosphate links that form a backbone from which the bases protrude.

5¿–3¿

LEWIMC01_001-022hr3 7/2/05 10:57 AM Page 5

6 CHAPTER 1 DNA Is the Hereditary Material

• A stretch of overwound DNA has more base pairs per turn thanthe usual average This means that the twostrands of DNA are more tightly wound around each other, cre-ating tension.

• A stretch of underwound DNA has fewer base pairs per turn thanthe usual average This means that the twostrands of DNA are less tightly wound around each other; ulti-mately this can lead to strand separation.

Key Concepts• The B-form of DNA is a double helix consisting of two polynu-

cleotide chains that run antiparallel.

• The nitrogenous bases of each chain are flat purine or pyrimidinerings that face inwards and pair with one another by hydrogenbonding to form A-T or G-C pairs only.

• The diameter of the double helix is 20 Å, and there is a completeturn every 34 Å, with 10 base pairs per turn.

• The double helix forms a major (wide) groove and a minor (narrow)groove.

Three notions converged in the construction of the double helix modelfor DNA by Watson and Crick in 1953:

• X-ray diffraction data showed that DNA has the form of a regu-lar helix, making a complete turn every 34 Å (3.4 nm), with a di-ameter of (2 nm). Since the distance between adjacentnucleotides is 3.4 Å, there must be 10 nucleotides per turn.

• The density of DNA suggests that the helix must contain twopolynucleotide chains. The constant diameter of the helix can beexplained if the bases in each chain face inward and arerestricted so that a purine is always opposite a pyrimidine, avoid-ing partnerships of purine-purine (too wide) or pyrimidine-pyrimidine (too narrow).

• Irrespective of the absolute amounts of each base, the proportionof G is always the same as the proportion of C in DNA, and theproportion of A is always the same as that of T.

Watson and Crick proposed that the two polynucleotide chains inthe double helix associate by hydrogen bonding between the nitroge-nous bases. G can hydrogen bond specifically only with C, while Acan bond specifically only with T. These reactions are described asbase pairing, and the paired bases (G with C, or A with T) are said tobe complementary.

The Watson–Crick model proposed that the two polynucleotidechains run in opposite directions (antiparallel), as illustrated inFigure 1.8. One strand runs in the direction, while its partnerruns

The sugar-phosphate backbone is on the outside and carries nega-tive charges on the phosphate groups.When DNA is in solution in vitro,the charges are neutralized by the binding of metal ions, typically by

In the cell, positively charged proteins provide some of the neu-tralizing force.These proteins play an important role in determining theorganization of DNA in the cell.

The bases are flat structures, lying in pairs perpendicular to the axisof the helix, inside the backbone. The double helix is often likened to a

Na+.

3¿ : 5¿.5¿ : 3¿

'20 Å

(10 bp = 1 turn).

(10 bp = 1 turn).

LEWIMC01_001-022hr3 7/2/05 10:57 AM Page 6

The double helix has constant width

Interior is hydrophobicPhosphates havenegative charges

Hydrogen bond

CH2

O O

CH2

P O

O

�O

O

P

O

O

3�

O

N

CHC

C

O

O

H

C

N

CH3

CHC

N

N N

N

N H

C

C

C

NHH

H

N

CHC

C

OC

N

NC N

NH

H

H

CH2

O

O

P O�

O O

O

O

P O�

O

CH2

O

O

O

P O�

O

CH2

O

O

O

P O�

O

N

CHC

C

O

O

H

C

N

CH3

CHC

N

N N

NC

C

C

N

N

H

HH

H

HC

C

CO

N

CN

N

H

H H

O

O

P O�O

�O

O

O

P O�O

CH2

O

5�

3�

5�

Figure 1.8 The double helix maintains a constant width because purines always facepyrimidines in the complementary A-T and G-C base pairs. The sequence in the figure isT-A, C-G, A-T, G-C.

DNA Is a Double Helix SECTION 1.6 7

spiral staircase: the base pairs form the treads, as illustrated schemati-cally in Figure 1.9. Proceeding along the helix, bases are stacked aboveone another like a pile of plates.

Each base pair is rotated around the axis of the helix relativeto the next base pair. So base pairs make a complete turn of 360°.The twisting of the two strands around one another forms a doublehelix with a minor groove ( across) and a major groove (across), as can be seen from the scale model of Figure 1.10. The doublehelix is right-handed; the turns run clockwise as viewed along the heli-cal axis.These features represent the accepted model for what is knownas the B-form of DNA.

It is important to realize that the B-form represents an average, nota precisely specified structure. DNA structure can change locally. If adouble helix has more base pairs per turn it is said to be overwound; ifit has fewer base pairs per turn it is underwound. Local winding can beaffected by the overall conformation of the DNA double helix in spaceor by the binding of proteins to specific sites.

'22 Å'12 Å

'10'36°

G

CT

G

C

3�

5�Sugar

Base

Phosphate

5�

3�

Flat base pairs connect the DNA strands

A

C

G

Figure 1.9 Flat base pairs lie perpendicular to the sugar-phosphate backbone.

The DNA double helix has two grooves

Major groove22 Å � across

Diameter � 20 Å

Per helicalturn � 34 Å

Minor groove12 Å � across

Figure 1.10 The two strands of DNA form a double helix.

LEWIMC01_001-022hr3 7/2/05 10:57 AM Page 7

8 CHAPTER 1 DNA Is the Hereditary Material

1.7 Supercoiling Affects theStructure of DNA

Key Term• Supercoiling describes the coiling of a double helix in which nei-

ther strand has a free end or any breaks; supercoiling causes theclosed double helix to cross over its own axis.

Key Concepts• Supercoiling occurs only in a closed DNA with no free ends.

• A closed DNA can be a circular DNA molecule or a linear moleculewith both ends anchored in a protein structure.

The winding of the two strands of DNA around each other in the doublehelical structure makes it possible to change the structure by modifyingits conformation in space. If the two ends of a DNA molecule are fixed,the double helix can be wound around itself. This is called supercoiling.The effect is like that of a rubber band twisted around itself.The simplestexample of a DNA with no fixed ends is a circular molecule.The effect ofsupercoiling can be seen in Figure 1.11 by comparing the nonsupercoiledcircular DNA lying flat with the supercoiled circular molecule that formsa twisted and therefore more condensed shape.

The consequences of supercoiling depend on whether the DNA istwisted around itself in the same direction as the two strands within thedouble helix (clockwise) or in the opposite direction. Twisting in thesame direction produces positive supercoiling.This has the effect of caus-ing the DNA strands to wind around one another more tightly, so thereare more base pairs per turn.Twisting in the opposite direction producesnegative supercoiling. This causes the DNA strands to be twisted aroundone another less tightly, so there are fewer base pairs per turn. Negativesupercoiling can be thought of as creating tension in the DNA that is re-lieved by unwinding the double helix. The ultimate effect of negative su-percoiling is to generate a region in which the two strands of DNA haveseparated—that has, in other words, zero base pairs per turn.

Topological manipulation of DNA is part of all of its functions—recombination, replication, and transcription—and of its higher-orderstructure. DNA synthesis requires the double strands to separate. How-ever, because the strands are intertwined, they must rotate about eachother to separate. Some possibilities for the unwinding reaction are il-lustrated in Figure 1.12.

We might envisage the structure of DNA in terms of a free end thatwould allow the strands to rotate about the axis of the double helix forunwinding. Given the length of the double helix, however, this wouldinvolve the separating strands in a considerable amount of flailingabout, which seems unlikely in the confines of the cell.

A similar result is achieved by placing an apparatus to control therotation at the free end. However, the effect must be transmitted over aconsiderable distance, again involving the rotation of an unreasonablelength of material.

Consider the effects of separating the two strands in a moleculewhose ends are not free to rotate. When two intertwined strands arepulled apart from one end, the result is to increase their winding abouteach other farther along the molecule. The problem can be overcomeby introducing a transient nick in one strand. An internal free end al-lows the nicked strand to rotate about the intact strand, after which thenick can be sealed. Each repetition of the nicking and sealing reactionreleases one superhelical turn.

Strand separation requires changes in topology

Rotation about a free end

Rotation at fixed ends

Nicking, rotation, and ligationNick

Strand separation compensated bypositive supercoiling

Figure 1.12 Separation of the strands of a DNA doublehelix could be achieved by several means.

Circular DNA can be supercoiled

Figure 1.11 Linear DNA is extended, a circular DNA re-mains extended if it is relaxed (nonsupercoiled), but a su-percoiled DNA has a twisted and condensed form.

LEWIMC01_001-022hr3 7/2/05 10:57 AM Page 8

Base pairing accounts for specificityof replication

Daughterstrands

Parentalstrand

unwinds

A

G

C

C

G

A

C

T

T

T

GC

A T

CG

G

AT

A

A

G

CG

A

C

T

T

Figure 1.13 Base pairing provides the mechanism forreplicating DNA.

The Structure of DNA Allows Replication and Transcription SECTION 1.8 9

Supercoiling can be measured as the density of supercoils per unitlength of DNA, which is called We will see that the supercoiling den-sity has important effects in vivo, and that specific enzymes are neces-sary to change it when the structure of DNA requires manipulation.

1.8 The Structure of DNA AllowsReplication and Transcription

Key Terms• A parental strand or duplex of DNA is the DNA that is replicated.

• The template strand (antisense strand) of DNA is complementaryto the sense strand, and is the one that acts as the template forsynthesis of mRNA.

• A daughter strand or duplex of DNA is the newly synthesized DNA.

• A DNA polymerase is an enzyme that synthesizes daughterstrands of DNA (under direction from a DNA template). Any partic-ular enzyme may be involved in repair or replication (or both).

• RNA polymerases are enzymes that synthesize RNA using a DNAtemplate (formally described as DNA-dependent RNA polymerases).

• Reverse transcriptase is an enzyme that uses a template ofsingle-stranded RNA to generate a double-stranded DNA copy.

Key Concepts• A template strand of a nucleic acid directs synthesis of a comple-

mentary product strand by base pairing.

• Nucleic acids are synthesized by adding nucleotides one by oneto the end of a polynucleotide chain.

• DNA polymerases use these reactions to synthesize DNA, andRNA polymerases use them to synthesize RNA.

It is crucial that the genetic material is reproduced accurately. Becausethe two polynucleotide strands are joined only by hydrogen bonds, theyare able to separate without requiring breakage of covalent bonds. Thespecificity of base pairing suggests that each of the separated parentalstrands could act as a template strand for the synthesis of a comple-mentary daughter strand. Figure 1.13 shows the principle that a newdaughter strand is assembled on each parental strand. The sequence ofthe daughter strand is dictated by the parental strand; an A in theparental strand causes a T to be placed in the daughter strand, aparental G directs incorporation of a daughter C, and so on.

The top part of the figure shows a parental (unreplicated) duplexthat consists of the original two parental strands. The lower part showsthe two daughter duplexes that are being produced by complementarybase pairing. Each of the daughter duplexes is identical in sequencewith the original parent, and contains one parental strand and onenewly synthesized strand. The structure of DNA carries the informationneeded to perpetuate its sequence.

The same principle is used in all nucleic acid synthesis:a specific enzymerecognizes the template and undertakes the task of catalyzing the addition ofsubunits to a polynucleotide chain that is being synthesized.The enzymes arenamed according to the type of chain that is synthesized. Figure 1.14 sum-marizes the types of enzymes and their substrates and products:

• DNA polymerase is responsible for the replication of double-stranded DNA. Each of the strands of the parental duplex acts as a

3¿ ¬ OH

s.

Polymerases synthesize nucleic acids

DNA polymerase replicates dsDNA

RNA polymerase transcribes dsDNA into ssRNA

Reverse transcriptase synthesizesdsDNA on ssRNA

Daughter duplexes are identical to parental duplex

RNA is identical to parental green strand

Figure 1.14 All nucleic acids are synthesized by poly-merases that use base pairing to ensure that each newchain is complementary to its template chain.

LEWIMC01_001-022hr3 7/2/05 10:57 AM Page 9

10 CHAPTER 1 DNA Is the Hereditary Material

5� end 5� end

Nucleic acids grow by extension of the 3� end

OCH2

�O

O

P

O

O

CH3

O

O

�O

O

P

O

O

CH2

OCH2

�O

O

P

O

O

CH3

O

O

�O

O

P

O

O

CH2

Figure 1.15 A nucleic acid chain extends from a end toa end. A new nucleotide has a triphosphate.When it is added to the chain, it breaks the bond to its ter-minal two phosphates, and the remaining phosphate islinked to the oxygen of the terminus of the chain.3¿ ¬ OH

5¿3¿ ¬ OH5¿

template to synthesize a complementary daughter strand. The re-sult is to produce two duplex DNAs, each of which is identical tothe original duplex.

• RNA polymerase (or more properly, DNA-dependent RNA poly-merase) copies a template strand of DNA into a complementaryRNA.The RNA is identical in sequence to the other (nontemplate)strand of DNA. The process is called transcription. DNA structureis disrupted only transiently, and the RNA product is released as asingle-stranded molecule.

• Reverse transcriptase can copy a template of single-strandedRNA into a complementary DNA strand. The reaction producesa double-stranded nucleic acid with one DNA strand and oneRNA strand. A similar reaction is catalyzed by RNA replicases(more properly RNA-dependent RNA polymerases) that are re-sponsible for replicating single-stranded RNA viruses.

The chemical reaction that synthesizes a nucleic acid is the same inall cases. Nucleotides are added one by one to the end of the chain.Figure 1.15 shows that an incoming nucleotide has a triphosphate

3¿

LEWIMC01_001-022hr3 7/2/05 10:57 AM Page 10

DNA Replication Is Semiconservative SECTION 1.9 11

group at its position. It reacts with the hydroxyl at the endof the polynucleotide chain. The two terminal phosphates are released,and a new bond is formed between the remaining phosphate and theoxygen atom of the terminal hydroxyl group. The polynucleotidechain is now one subunit longer.

1.9 DNA Replication IsSemiconservative

Key Term• Semiconservative replication is accomplished by separation of

the strands of a parental duplex, each then acting as a templatefor synthesis of a complementary strand.

Key Concepts• The Meselson-Stahl experiment used density labeling to prove

that the single polynucleotide strand is the unit of DNA that isconserved during replication.

• Each strand of a DNA duplex acts as a template to synthesize adaughter strand.

• The sequences of the daughter strands are determined by com-plementary base pairing with the separated parental strands.

A parental duplex of DNA replicates to form two daughter duplexes,each of which consists of one parental strand and one (newly synthe-sized) daughter strand.The units that are conserved from one generationto the next are the two individual strands of the parental duplex. This be-havior is called semiconservative replication.

Experimental support for the model of semiconservative replica-tion is illustrated in Figure 1.16. If a parental DNA carries a “heavy”density label because the organism has been grown inmedium containing a suitable isotope (such as ), itsstrands can be distinguished from those that are synthe-sized when the organism is transferred to a medium con-taining normal “light” isotopes.

The parental DNA consists of a duplex of two heavystrands (red). After one generation of growth in lightmedium, the duplex DNA is “hybrid” in density—it con-sists of one heavy parental strand (red) and one lightdaughter strand (blue). After a second generation, thetwo strands of each hybrid duplex have separated; eachgains a light partner, so that now half of the duplex DNAremains hybrid while half is entirely light (both strandsare blue).

This experimental confirmation demonstrating thatthe individual strands of these duplexes are entirely heavyor entirely light is the Meselson-Stahl experiment of 1958,which followed the semiconservative replication of DNAthrough three generations of growth of E. coli. WhenDNA was extracted from bacteria and its density mea-sured by centrifugation, the DNA formed bands corre-sponding to its density—heavy for parental, hybrid for thefirst generation, and half hybrid and half light in the sec-ond generation.

15N

3¿

3¿ ¬ OH5¿

DNA single strands are the conserved units

Generation 1Parental DNA

Density analysis

Heavy

Hybrid

Hybrid

Hybrid

Light

Light

– Heavy– Hybrid– Light

– Heavy– Hybrid– Light

– Heavy– Hybrid– Light

Hybrid

Replicate ina light density medium

Generation 2

Figure 1.16 Replication of DNA is semiconservative.

LEWIMC01_001-022hr3 7/2/05 10:57 AM Page 11

Endonucleases attack internal bonds

Broken bond

Figure 1.18 An endonuclease cleaves a bond within a nu-cleic acid. This example shows an enzyme that attacks onestrand of a DNA duplex.

12 CHAPTER 1 DNA Is the Hereditary Material

A replication fork moves along DNA

Parental DNA

Replication fork

Replicated DNA

Figure 1.17 The replication fork is the region of DNA inwhich there is a transition from the unwound parental du-plex to the newly replicated daughter duplexes.

1.10 DNA Strands Separateat the Replication Fork

Key Terms• A replication fork (growing point) is the

point at which strands of parental duplexDNA separate so that replication canproceed. A complex of proteins includingDNA polymerase interact at the fork.

• A deoxyribonuclease (DNAase) is an en-zyme that specifically attacks bonds inDNA. It may cut only one strand or both.

• Ribonucleases (RNAase) are enzymes thatcleave RNA. They may be specific forsingle-stranded or for double-strandedRNA, and may be either endonucleases orexonucleases.

• Exonucleases cleave nucleotides one ata time from the end of a polynucleotidechain; they may be specific for either the

or end of DNA or RNA.• Endonucleases cleave bonds within a nu-

cleic acid chain; they may be specific forRNA or for single-stranded or double-stranded DNA.

3¿5¿

Key Concept• Replication of DNA is accomplished by a complex of enzymes

that separate the parental strands at the replication fork and syn-thesize the daughter strands.

Replication requires the two strands of the parental duplex to separate.This disruption of structure is only transient, however, and is reversedas the daughter duplex forms. Only a small stretch of the duplex DNAis separated into single strands at any moment.

The helical structure of a molecule of DNA engaged in replicationis illustrated in Figure 1.17. The nonreplicated region consists of theparental duplex, opening into the replicated region where the twodaughter duplexes have formed.The double helical structure is disrupt-ed at the junction between the two regions, which is called thereplication fork. As the DNA replicates, the replication fork movesalong the parental DNA, so there is a continuous unwinding of theparental strands and rewinding into daughter duplexes.

Degradation of nucleic acids also requires specific enzymes:deoxyribonucleases (DNAases) degrade DNA, and ribonucleases(RNAases) degrade RNA. The nucleases fall into the general classes ofexonucleases and endonucleases:

• Endonucleases cut individual bonds within RNA or DNA mole-cules, generating discrete fragments. Some DNAases cleave bothstrands of a duplex DNA at the target site, while others cleave onlyone of the two strands. Endonucleases catalyze cutting reactions, asshown in Figure 1.18.

• Exonucleases remove residues one at a time from the end of a mol-ecule, generating mononucleotides. They always function on a sin-gle nucleic acid strand, and each exonuclease proceeds in a specificdirection, that is, starting at either a or a end and proceedingtoward the other end. They catalyze trimming reactions, as shownin Figure 1.19.

1.11 Genetic Information Can beProvided by DNA or RNA

Key Term• The central dogma describes the basic nature of genetic infor-

mation: sequences of nucleic acid can be perpetuated and inter-converted by replication, transcription, and reverse transcription,but translation from nucleic acid to protein is unidirectional, be-cause nucleic acid sequences cannot be retrieved from proteinsequences.

Key Concepts• Cellular genes are DNA, but viruses and viroids may have genes

of RNA.

• DNA is converted into RNA by transcription, and RNA may beconverted into DNA by reverse transcription.

• The translation of RNA into protein is unidirectional.

3¿5¿

LEWIMC01_001-022hr3 7/2/05 10:57 AM Page 12

The central dogma describes information flow

Protein

Translation

Replication

DNA

DNA

RNA

TranscriptionReversetranscription

Figure 1.20 The central dogma states that information innucleic acid can be perpetuated or transferred, but thetransfer of information into protein is irreversible.

Exonucleases nibble from the ends

Figure 1.19 An exonuclease removes bases one at a timeby cleaving the last bond in a polynucleotide chain.

Genetic Information Can be Provided by DNA or RNA SECTION 1.11 13

The central dogma defines the paradigm of molecular biology. Genesare perpetuated as sequences of nucleic acid, but function by being ex-pressed in the form of proteins. Replication of DNA is responsible forthe inheritance of genetic information. Transcription of DNA intoRNA and translation of RNA into protein are responsible for the con-version from one form of genetic information to another.

Figure 1.20 illustrates the roles of replication, transcription, andtranslation, viewed from the perspective of the central dogma:

• The perpetuation of nucleic acid may use either DNA or RNA as thegenetic material. Cells use only DNA. Some viruses use RNA, andreplication of viral RNA occurs in an infected cell.

• The expression of cellular genetic information usually is unidirec-tional. Transcription of DNA generates RNA molecules that can beused further only to generate protein sequences; generally theycannot be retrieved for use as genetic information. Translation ofRNA into protein is always irreversible.

Replication, transcription, and translation maintain and expressthe cellular genetic information of prokaryotes or eukaryotes, and theinformation carried by viruses. The genomes of all living organismsconsist of duplex DNA. Viruses have genomes that consist of DNA orRNA, and there are examples of each type that are double-stranded(ds) or single-stranded (ss). The general principle of the nature of thegenetic material, then, is that it is always nucleic acid; in fact, it is DNAexcept in the RNA viruses. Details of the mechanism used to replicatethe nucleic acid vary among the viral systems, but the principle of repli-cation via synthesis of complementary strands remains the same, as il-lustrated in Figure 1.21.

Cellular genomes reproduce DNA by the mechanism of semicon-servative replication. Double-stranded viral genomes, whether DNA orRNA, also replicate by using the individual strands of the duplex astemplates to synthesize partner strands.

Viruses with single-stranded genomes use the single strand as tem-plate to synthesize a complementary strand; and this complementarystrand in turn is used to synthesize its complement, which is, of course,identical with the original starting strand. Replication may in-volve the formation of stable double-stranded intermediatesor use double-stranded nucleic acid only as a transient stage.

The usual direction for information transfer is from DNAto RNA. However, this is reversed in the retroviruses, whosegenomes consist of single-stranded RNA molecules. Duringthe infective cycle, the RNA is converted by the process ofreverse transcription into a single-stranded DNA, which inturn is converted into a double-stranded DNA. This duplexDNA becomes part of the genome of the cell, and is inheritedlike any other gene. So reverse transcription allows a sequenceof RNA to be retrieved and used as genetic information.

The existence of RNA replication and reverse transcrip-tion establishes the general principle that information in theform of either type of nucleic acid sequence can be convertedinto the other type. In the usual course of events, however,the cell relies on the processes of DNA replication, tran-scription, and translation. But on rare occasions (possiblymediated by an RNA virus), information from a cellularRNA is converted into DNA and inserted into the genome.Although reverse transcription plays no role in the regularoperations of the cell, it becomes a mechanism of potentialimportance when we consider the evolution of the genome.

Nucleic acids replicate via complementary strands

Replication generates two daughter duplexes each containingone parental strand and one newly synthesized strand

Single parental strandis used to synthesizecomplementary strand

Complementary strandis used to synthesize a copyof the parental strand

Old strand

Old strand

New strands

Double-strandedtemplate

Single-strandedtemplate

Old strandNew strand

Figure 1.21 Double-stranded and single-stranded nucleic acids bothreplicate by synthesis of complementary strands governed by the rulesof base pairing.

LEWIMC01_001-022hr3 7/2/05 10:57 AM Page 13

Both DNA and RNA may form duplexes

DNA

Intramolecularpairing withinRNA

Intramolecularpairing betweenshort and longRNAs

Figure 1.22 Base pairing occurs in duplex DNA and also inintra- and intermolecular interactions in single-strandedRNA (or DNA).

14 CHAPTER 1 DNA Is the Hereditary Material

1.12 Nucleic Acids Hybridizeby Base Pairing

Key Terms• Denaturation of DNA or RNA describes

its conversion from the double-strandedto the single-stranded state; usuallystrand separation is caused by heating.

• Renaturation describes the reassocia-tion of denatured complementary singlestrands of a DNA double helix.

• Annealing of DNA describes the renatu-ration of a duplex structure from singlestrands that were obtained by denaturingduplex DNA.

• Hybridization describes the pairing ofcomplementary RNA and DNA strands togive an RNA-DNA hybrid.

Key Concepts• Heating causes the two strands of a DNA duplex to separate.• The is the midpoint of the temperature range for denaturation.• Complementary single strands can renature when the temperature

is reduced.• Denaturation and renaturation/hybridization can occur with DNA-

DNA, DNA-RNA, or RNA-RNA combinations, and can be inter-molecular or intramolecular.

• The ability of two single-stranded nucleic acid preparations tohybridize is a measure of their complementarity.

The concept of base pairing is central to all processes involving nucleicacids. Disruption of the base pairs is a crucial aspect of the function of adouble-stranded molecule, while the ability to form base pairs is essentialfor the activity of a single-stranded nucleic acid.

A crucial property of the double helix is the ability to separate thetwo strands without disrupting covalent bonds. This makes it possiblefor the strands to separate and reform under physiological conditionsat the (very rapid) rates needed to sustain genetic functions. The speci-ficity of reformation is determined by complementary base pairing.

Formation of duplex regions from single-stranded nucleic acids ismost important for RNA. Figure 1.22 shows that base pairing enablescomplementary single-stranded nucleic acids to form a duplex struc-ture. This can be either intramolecular or intermolecular:

• An intramolecular duplex region can form by base pairing betweentwo complementary sequences at different positions along a single-stranded molecule.

• A single-stranded molecule may base pair with an independent,complementary single-stranded molecule to form an intermolecu-lar duplex.

The lack of covalent links between complementary strands makesit possible to manipulate DNA in vitro.The noncovalent forces that sta-bilize the double helix are disrupted by heating or by exposure to lowsalt concentration. The two strands of a double helix separate entirelywhen all the hydrogen bonds between them are broken.

The process of separating the strands of DNA is called denaturat-ion or (more colloquially) melting. (“Denaturation” is not restricted todescribing changes in DNA, but is also used more generally to describeloss of authentic structure in any situation where the natural conforma-tion of a macromolecule has been converted to some other form.)

DNA denatures over a narrow temperature range, bringing aboutstriking changes in many of its physical properties. The midpoint of thetemperature range over which the strands of DNA separate is calledthe melting temperature It depends on the proportion of base pairs. Because each base pair has three hydrogen bonds, it ismore stable than an base pair, which has only two hydrogenbonds. The more base pairs are contained in a DNA, the greaterthe energy that is needed to separate the two strands. In solution underphysiological conditions, a DNA that is 40% —a value typical ofmammalian genomes—denatures with a of about 87°C. So duplexDNA is stable at the temperature prevailing in the cell.

The denaturation of DNA is reversible under appropriate conditions.The ability of the two separated complementary strands to reform into a

Tm

G # CG # C

A # TG # C

G # C1Tm2.

Tm

LEWIMC01_001-022hr3 7/2/05 10:57 AM Page 14

The filter assay measurescomplementarity

Figure 1.24 Filter hybridization establishes whether a so-lution of denatured DNA (or RNA) contains sequences com-plementary to the strands immobilized on the filter.

DNA can be denatured and renatured

Single-stranded DNA

Double-stranded DNA

Renatured DNA

Renaturation

Denaturation

Figure 1.23 Denatured single strands of DNA can renatureto give the duplex form.

Mutations Change the Sequence of DNA SECTION 1.13 15

double helix is called renaturation. Renaturation depends on specific basepairing between the complementary strands. Figure 1.23 shows that the re-action takes place in two stages. First, single strands of DNA in the solutionencounter one another by chance; if their sequences are complementary,the two strands base pair to generate a short double-helical region.Then theregion of base pairing extends along the molecule by a zipper-like effect toform a lengthy duplex molecule. Renaturation of the double helix restoresthe original properties that were lost when the DNA was denatured.

Renaturation describes the reaction between two complementary se-quences that were separated by denaturation. However, the technique canbe extended to allow any two complementary nucleic acid sequences toreact with each other to form a duplex structure. This is sometimes calledannealing, but the reaction is more generally described as hybridizationwhenever nucleic acids of different sources are involved, as in the casewhen one preparation consists of DNA and the other consists of RNA.

The principle of the hybridization reaction is to expose two single-stranded nucleic acid preparations to each other and then to measurethe amount of double-stranded material that forms. Figure 1.24 illus-trates a procedure in which a DNA preparation is denatured and thesingle strands are adsorbed to a filter. Then the filter is immersed in asecond denatured DNA (or RNA) preparation. The filter has beentreated so that the second preparation can adsorb to it only if it is ableto base pair with the DNA that was originally adsorbed. Usually thesecond preparation is radioactively labeled, so that the reaction can bemeasured as the amount of radioactive label retained by the filter.

The extent of hybridization between two single-stranded nucleic acidsconstitutes a precise measure of their complementarity. Two sequences neednot be perfectly complementary to hybridize. If they are closely related butnot identical, an imperfect duplex is formed in which base pairing is inter-rupted at positions where the two single strands do not correspond.

1.13 Mutations Changethe Sequence of DNA

Key Terms• A mutation is any change in the sequence of genomic DNA.• Spontaneous mutations occur in the absence of any added

reagent to increase the mutation rate, as the result of errors in repli-cation (or other events involved in the reproduction of DNA) or byenvironmental damage.

• The background level of mutation describes the rate at which se-quence changes accumulate in the genome of an organism. It reflectsthe balance between the occurrence of spontaneous mutations andtheir removal by repair systems, and is characteristic for any species.

• Mutagens increase the rate of mutation by inducing changes inDNA sequence, directly or indirectly.

• Induced mutations result from the action of a mutagen. The mu-tagen may act directly on the bases in DNA or it may act indirect-ly to trigger a pathway that leads to a change in DNA sequence.

Key Concepts• Mutations provide the basis for evolution.• Mutations may occur spontaneously or may be induced by mutagens.• The rate of mutation in any organism depends on the balance be-

tween the occurrence of mutations and their removal by cellularsystems.

LEWIMC01_001-022hr3 7/2/05 10:57 AM Page 15

16 CHAPTER 1 DNA Is the Hereditary Material

Mutation rates increase with target size

Mutation rate Any base pair1 in 109�1010

Any gene1 in 105�106

generations

...AGCTGTCATGGGTACATTA...

...TCGACAGTACCCATGTAAT...

The genome1 in 300generations

CHC

N

N N

NC

C

C

N

N

H H

H

H

CN N

CHCO

C

C

CH3

O

Figure 1.25 On the average, a base pair mutates at a rateof per generation, a gene of 1000 bp mutatesat per generation, and a bacterial genome mutatesat per generation.3 * 10-3

'10-610-9–10-10

The sequence of DNA determines the nature of any organism. Differ-ences between the genetic properties of the individual members of aspecies are the consequence of differences in their DNA sequences.Any change that is made in the sequence of a genome from one gener-ation to the next is called a mutation.

Not all mutations affect the phenotype of an organism, but any mu-tation with phenotypic consequences means that individuals with themutation differ in some way from individuals who do not have it. Thisprovides the basis for evolution to select among individuals.

Most mutations that influence the phenotype do so by changing theproperties or the amount of some protein. When a change in the se-quence of DNA causes an alteration in the sequence of a protein, wemay conclude that the DNA codes for that protein. The existence ofmany mutations in a gene may allow many variant forms of a protein tobe compared, and a detailed analysis can be used to identify regions ofthe protein responsible for individual enzymatic or other functions.

All organisms suffer a certain number of mutations as the result of nor-mal cellular operations or random interactions with the environment.Theseare called spontaneous mutations; the rate at which they occur is character-istic for any particular organism and is sometimes called the backgroundlevel. Mutations are rare events, and those that damage a favorable pheno-type are selected against during evolution. It is therefore difficult to obtainlarge numbers of spontaneous mutants to study from natural populations.

The occurrence of mutations can be increased by treatment withcertain compounds. These are called mutagens, and the changes theycause are referred to as induced mutations. Most mutagens act directlyeither by modifying a particular base of DNA or by becoming incorpo-rated into the nucleic acid. The effectiveness of a mutagen is judged byhow much it increases the rate of mutation above background. By usingmutagens, it becomes possible to induce many changes in any gene.

When a mutation occurs in a somatic cell, it can affect only the indi-vidual carrying that cell and other cells descended from it.When a muta-tion occurs in the germline, it may be inherited by the next generation.However, all organisms have cellular systems that counteract mutationsby attempting to correct changes in DNA. The rate at which mutationsaccumulate depends on the balance between the rate at which they occurand the efficiency of the cellular system in removing them.

Spontaneous mutations that inactivate gene function accumulate inbacteriophages and bacteria at a relatively constant rate of per genome per generation. Figure 1.25 shows that in bacteria the muta-tion rate corresponds to events per gene per generation or to anaverage rate of change per base pair of per generation. Themutation rate of individual base pairs varies very widely, however, over a10,000 fold range.We have no accurate measurement of the rate of muta-tion in eukaryotes, although usually it is thought to be somewhat similarto that of bacteria on a per-locus per-generation basis.

1.14 Mutations May Affect SingleBase Pairs or LongerSequences

Key Terms• A point mutation is a change in the sequence of DNA involving a

single base pair.

• A transition is a mutation in which one pyrimidine is replaced bythe other or in which one purine is replaced by the other.

10-9–10-10'10-6

3–4 * 10-3

LEWIMC01_001-022hr3 7/2/05 10:57 AM Page 16

Mutations May Affect Single Base Pairs or Longer Sequences SECTION 1.14 17

• A transversion is a mutation in which a purine is replaced by apyrimidine or vice versa.

• Base mispairing is a coupling between two bases that does notconform to the Watson-Crick rule, e.g., adenine with cytosine,thymine with guanine.

• An insertion is the addition of a stretch of base pairs in DNA. Du-plications are a special class of insertions.

• A transposon (transposable element) is a DNA sequence able toinsert itself (or a copy of itself) at a new location in the genome,without having any sequence relationship with the target locus.

• A deletion is the removal of a sequence of DNA, the regions oneither side being joined together except in the case of a terminaldeletion at the end of a chromosome.

Key Concepts• Point mutations can be caused by the chemical conversion of one

base into another or by mistakes that occur during replication.• Insertions are the most common type of mutation, and result from

the movement of transposable elements.

Mutations can take various forms, ranging from insertions or deletionsof large amounts of DNA to changes in single base pairs.

Any base pair of DNA can be mutated. A point mutation changesonly a single base pair, and can be caused by either of two types of event:

• Chemical modification of DNA directly changes one base into adifferent base.

• A malfunction during the replication of DNA causes the wrongbase to be inserted into a polynucleotide chain during DNAsynthesis.Point mutations can be divided into two classes, depending on the

type of change by which one base is substituted for another:• The more common class is the transition, comprising the substitu-

tion of one pyrimidine by the other, or of one purine by the other.This replaces a pair with an pair or vice versa.

• The less common class is the transversion, in which a purine is re-placed by a pyrimidine or vice versa, so that an pair becomesa or pair.The effects of nitrous acid provide a classic example of a transition

caused by the chemical conversion of one base into another. Figure 1.26shows that nitrous acid performs an oxidative deamination that convertscytosine into uracil. In the replication cycle following the transition, theU pairs with an A, instead of with the G with which the original C wouldhave paired. So the pair is replaced by a pair when the Apairs with the T in the next replication cycle. (Nitrous acid also deami-nates adenine, causing the reverse transition from to )

Transitions are also caused by base mispairing, when unusual part-ners pair in defiance of the usual restriction to Watson-Crick pairs. Basemispairing usually results from the incorporation into DNA of an ab-normal base that has ambiguous pairing properties. Figure 1.27 showsthe example of bromouracil (BrdU), an analog of thymine that containsa bromine atom in place of the methyl group of thymine. BrdU is incor-porated into DNA in place of thymine. But it has ambiguous pairingproperties because the presence of the bromine atom allows a shift inwhich the base changes structure from a keto form to an enol

form. The enol form can base pair with guanine, which leadsto substitution of the original pair by a pair.G # CA # T1¬ OH2

1“ O2

G # C.A # TT # AC # G

C # GT # AA # T

A # TG # C

BrdU causes A-T to be replaced by G-C

Keto-enol shift allows BrdU to pair with G

Keto-enolshift

T-A pair

BrdU pairs withA at replication

BrdU pairs withG at replication

Backbone

Backbone

CH3

H

BrdU-A pair

BrdU-G pair

Backbone

N

H

N

C NN

CHNC

C

NH H

HC

NC

O

CC

N

HC

NC

CC

N

HC

NC

CC

N

HC

NC

CC

N

HC

O

H

H

Backbone

Backbone

BR

HN

C NN

CHNC

C

NH H

HCO

O

Backbone

N

C NN

CHNC

C

O

C

BR

H

O

O

BR

H

NC

O

CC

N

HCO

BRH

O

O

Figure 1.27 Mutations can be induced by the incorpora-tion of base analogs into DNA.

Nitrous acid deaminates cytosine to uracil

Nitrous acid

Cytosine

Replication

Mutant

Wild type

C

G

U

G

U

AC

G

Uracil

H

N

O

Figure 1.26 Mutations can be induced by chemical modifi-cation of a base.

LEWIMC01_001-022hr3 7/2/05 10:57 AM Page 17

Some mutations can revert

Point mutation

...ATCGGACTTACCGGTTA...

...TAGCCTGAATGGCCAAT...

Insertion

...ATCGGACTTACCGGTTA...

...TAGCCTGAATGGCCAAT...

Deletion

No reversion possible

...ATCGGACTTACCGGTTA...

...TAGCCTGAATGGCCAAT...

...ATCGGACGGTTA...

...TAGCCTGCCAAT...

...ATCGGACTTACCGGTTA...

...TAGCCTGAATGGCCAAT...

Reversion by deletion

...ATCGGACTTXXXXXACCGGTTA...

...TAGCCTGAAYYYYYTGGCCAAT...

...ATCGGACTTACCGGTTA...

...TAGCCTGAATGGCCAAT...

Reversion

...ATCGGACTCACCGGTTA...

...TAGCCTGAGTGGCCAAT...

Figure 1.28 Point mutations and insertions can revert, butdeletions cannot revert.

18 CHAPTER 1 DNA Is the Hereditary Material

The mispairing can occur either during the original incorporationof the base or in a subsequent replication cycle. The transition is in-duced with a certain probability in each replication cycle, so the incor-poration of BrdU has continuing effects on the sequence of DNA.

Point mutations were thought for a long time to be the principalmeans of change in individual genes. However, we now know thatinsertions of stretches of additional material are quite frequent. Thesource of the inserted material lies with transposons, sequences ofDNA with the ability to move from one site to another (see Chapter 21Transposons and Chapter 22 Retroviruses and Retroposons). An inser-tion usually abolishes the activity of a gene.Where such insertions haveoccurred, deletions of part or all of the inserted material, and some-times