Nucleases are a class of enzymes that catalyze the hydrolysis of nucleic acids (DNA, RNA) in all organisms, including humans. In addition to their important biological role, nucleases have recently emerged as useful tools in laboratory studies, and have led to the development of such fields as recombinant DNA technology, molecular cloning, and genomics. Nawin Mishra’s Nucleases, the first comprehensive treatment of the subject, introduces the properties and biological roles of nucleases to newcomers in the field and provides the basis of their possible application to critical aspects of science, commerce, and industry.
Covering the structure and biochemical properties of nucleases, Mishra’s text further guides the reader to understand how these enzymes can be exploited to make new products for the diagnosis and treatment of disease. Molecular cloning, made possible by the evolving understanding and application of nucleases, may well lead to the identification and characterization of genes responsible for diseases and their possible alleviation by gene therapy and the development of designer drugs. Nucleases is a one–stop, authoritative resource for students of biology and researchers currently practicing in the field. Chapter topics include:
- Restriction Endonucleases
- Sugar Non–Specific Nucleases
- Molecules That Interact with Nucleases
An ideal text and reference work for professionals and students in biochemistry, genetics, and molecular biology, Nucleases promises to be the field’s standard–bearer for years to come.
List of Nobel Prize Winners for Their Research Work with Nucleases.
About the Author.
I. Historical Perspectives.
II. Protein, RNA, DNA, and Other Molecules as Nucleases.
III. Nature of Enzymatic Reactions Catalyzed by Nucleases.
A. Nature of Substrates.
B. Mode of Attack.
C. Site–Specificity and Structure–Selectivity.
V. Methods for the Study of Nucleases.
A. Methods for the Assay of the Enzymatic Activity.
B. Methods for the Study and Characterization of Nucleases.
VI. Genetics of Nucleases and Biological Roles.
VII. Applications of Nucleases.
I. General Ribonucleases.
A. Microbial Ribonucleases.
B. Mammalian Ribonucleases.
1. Bovine Pancreatic Rnase.
3. Human Pancreatic Ribonuclease (HPR).
4. Human Nonsecretory Ribonuclease (HNSR).
5. Human Major Basic Protein (MBP), Eosinophil Cationic Protein (ECP) and Eosinophil–Derived Neurotoxin (EDN).
7. Interferon–Induced Mammalian Ribonuclease.
8. Human RNase with a Possible Role in Tumor Suppression.
C. Plant Ribonucleases.
D. Evolution of Ribonucleases.
II. Ribonucleases Involved in RNA Processing (Trimming, Splicing, and Editing).
A. RNaseIII and RNaseIII–Like Enzymes.
F. Eukaryotic RNA–Splicing Enzymes.
1. Yeast tRNA Splicing Endonuclease.
III. Ribonuclease H.
A. E. coli RNaseH.
B. Retroviral Reverse Transcriptase RNaseH.
C. Yeast RNaseH.
D. Human RNaseH.
E. Other Eukaryotic RNaseH.
F. Biological Function of RNaseH.
IV. Proofreading Activity of RNA Polymerase.
I. Classification of Enzymes.
II. Properties of Enzymes from Different Organisms.
A. Bacterial Enzymes.
1. Exonuclease I.
2. Exonuclease II.
3. Exonuclease III.
4. Application of the Enzyme Exonuclease III.
5. Exonucleases IVA and IVB.
6. Exonuclease V (RecBCD Enzyme).
7. RecBCD (Exo V) from Other Organisms.
8. Exonuclease VI.
9. Exonuclease VII.
10. Exonucleases Associated with DNA Polymerases.
11. Exonuclease VIII.
1. Bacterial Enzymes.
2. Mammalian Deoxyribonuclease.
4. Restriction Endonucleases.
I. Occurrence, Classification, and Their General Properties.
A. Different Restriction Endonucleases and Their Properties.
II. Type I Restriction Endonucleases.
A. Purification and General Properties.
B. Recognition Sequences and Nature of Substrate.
C. Different Kinds of Type II Restriction Endonucleases.
E. Cleavage Mechanism.
III. Type II Restriction Endonucleases.
A. Enzyme Purification and Assay.
B. General Properties of the Enzyme.
C. Crystal Structure of the Restriction Endonucleases.
D. Reaction Conditions and Enzyme Specificity.
E. Nature of Substrate.
1. Synthetic Oligonucleotides.
2. DNA with Base Analogs.
3. Methylated DNA.
4. Single–Stranded DNA.
5. DNA–RNA Hybrids as Substrate.
F. Inhibition of Restriction Endonucleases.
G. Restriction Endonuclease Genes.
IV. Type III Restriction Endonucleases.
V. Evolutionary Significance and Biological Role.
VI. Application of Restriction Nucleases.
VII. General Tips for Beginners or the First–Time Users of Restriction Enzymes.
5. Damage–Specific Nucleases.
I. Classification and Assay .
A. AP Endonucleases.
B. Enzymes that Directly Attack Phosphodiester Linkages in the Damaged DNA Region.
II. Properties of Two Groups of Enzymes from Different Organisms.
A. AP Endonucleases.
1. AP Endonucleases Associated with DNA Glycosylase Activity.
2. M. luteus Enzyme.
3. E. coli Endonuclease III.
B. AP Endonuclease Associated with Other Enzyme Activities.
1. E. coli Exonuclease III AP–Endonuclease Activity.
C. AP Endonucleases.
1. E. coli AP Endonucleases.
2. Fungal Apurinic Endonuclease.
3. Drosophila AP Endonucleases.
4. Human AP Endonucleases.
5. Plant AP Endonuclease.
D. Direct–Acting Enzymes.
1. E. coli UV Endonuclease.
2. Human Excision Nuclease.
I. Choreography and Topology of DNA.
II. Enzyme Assay.
A. Electron Microscopy.
B. Sedimentation Methods.
C. Agarose Gel Electrophoresis.
III. Properties of Enzymes from Different Groups of Organisms.
A. Prokaryotic Topoisomerases.
1. Prokaryotic Topoisomerase I.
2. Prokaryotic Topoisomerase II.
3. Properties of Gyrase.
4. Other Activities of Gyrase.
5. Prokaryotic Topoisomerase III.
B. Eukaryotic Topoisomerases.
1. Eukaryotic Topoisomerase I.
2. Eukaryotic Topoisomerase II.
C. Mitochondrial Topoisomerase.
D. Viral Topoisomerases.
IV. Genetics and Biological Role.
A. Prokaryotic Topoisomerase Mutants.
1. Topoisomerase I.
2. Topoisomerase II.
B. Eukaryotic Topoisomerase Mutants.
1. Topoisomerase I Mutants of Yeast.
2. Topoisomerase II Mutants of Yeast.
3. Topoisomerase Mutants of Higher Eukaryotes.
I. General Description and Classification.
A. General Recombinase.
B. Site–Specific Recombinase.
C. Transpositional Recombinase.
D. RNA Recombinase.
II. Properties of Different Recombinases.
A. General Recombinase.
B. Site–Specific Recombinase.
C. Prokaryotic Site–Specific Recombinase.
D. Eukaryotic Site–Specific Recombinase.
1. Eukaryotic Site–Specific Recombinase.
2. ′′Homing′′ Nuclease (Intron Coded Nuclease).
3. Viral Integrase.
E. Transpositional Recombinase.
1. Prokaryotic Transposases.
2. Eukaryotic Transposase.
3. Retrotransposable Elements and Retrotransposases.
F. Control of Recombinases.
G. RNA Recombinase.
8. Sugar–NonSpecific Nucleases.
I. General Description, Classification, and Methods of Assay.
II. Properties of Enzymes from Different Groups of Organisms.
A. Microbial Nucleases.
1. Neurospora crassa Endonuclease.
2. S1 Nuclease.
3. Yeast Nucleases.
4. Micrococcal Nuclease.
5. Bal–31 Nucleases.
B. Animal Nucleases.
1. Spleen Exonucleases.
2. Snake Venom Exonuclease.
C. Plant Nucleases.
1. Mung Bean Endonuclease.
2. Other Plant Nucleases.
D. Parasitic Protozoan Nuclease.
9. Nonprotein Nucleases.
1. Protein Component of RNaseP.
B. Introns as Ribozymes.
1. Group I Intron Ribozymes.
2. Mechanism of Catalysis by Group I Intron Ribozyme.
3. Assay of Ribozyme Activity of Intron RNA or Other RNA.
C. Group II Intron Ribozymes.
D. Splicosomal snRNA Ribozyme.
1. Proteins that Facilitate the Ribozyme Activity of RNA Nucleases.
F. Hammerhead RNA as Ribozyme.
G. Cis– and Trans–Acting Ribozyme Endonuclease.
A. Chemicals and Metal Ligand Complexes as Nucleases.
2. Hydrogen Peroxide.
3. DNA Intercalating Agents.
5. Factors Controlling the DNA Cleavage by Chemzymes.
IV. Designer Nuclease.
10. Molecules that Interact with Nucleases.
A. Proteins as Nuclease Inhibitors.
1. DNase Inhibitor–Protein.
2. RNase Inhibitor–Protein. B. RNA as Nuclease Inhibitors.
C. Other Molecules that Act as Nuclease Inhibitors.
II. Proteins that Interfere with the Activity of Nuclease by Interacting with the Substrate (Nucleic Acids).
III. DNA Sequences that Interact with Nucleases.
A. Chi–Like Elements in Eukaryotes.
IV. Other Inhibitor Molecules.
V. Proteins that Interact with DNA or Nuclease to Orchestrate the Activity of Nucleases.
11. Biological Function of Nucleases.
A. Three Steps in DNA Replication.
B. Role of Viral Nuclease in the Degradation of Host DNA.
C. Involvement of Nuclease During the Separation of Daughter Helices at the End of Replication.
D. Involvement of Nucleases in the Rolling Circle Mechanism of DNA Replication.
E. Involvement of Nuclease in the Replication of Linear DNA.
F. Involvement of Nuclease in the Replication of Chromosome in Eukaryotes.
II. DNA Repair.
A. Baseless Sites.
B. Sites with Altered Base or Incorrect Base.
C. Cross–Linking and Other Damages.
D. DNA Repair Mechanisms.
E. Excision Repair.
F. Bypass Repair Pathways.
G. Recombinational Repair Pathway.
H. Inducible and Error–Prone Repair Pathway.
I. Mismatch Repair.
J. Mismatch Repair in Mammalian Cells.
K. Incision of Damaged DNA is a Complex Process Involving Several Proteins.
L. Excision Repair Mutants of Neurospora.
M. Excision Repair Mutants of Yeast.
N. Excision Repair Mutants of Drosophila.
O. Excision Repair Mutants of Mammalian Cells.
A. Different Kinds of Genetic Recombination.
B. Recombination Mechanisms and Nucleases.
C. Gene Conversion and Postmeiotic Segregation.
D. In Vitro Recombination System.
E. Fungal Recombination Nucleases.
F. Mismatch Repairs During Recombination.
G. Recombination Pathways.
1. RecBCD Pathway.
2. RecFJ Pathway.
3. RecE Pathway.
4. Red Pathway.
H. Recombinational Control of Gene Expression.
I. Role of Recombinase in Mammalian Antibody Diversity, Allelic Exclusion, and Class Switch.
J. T–Cell Surface Receptor.
K. Application of Recombinases: Engineered Expression of Genes.
IV. DNA Transfection or Transformation.
VI. DNA Supercoiling and Maintenance of Chromosome Structure.
VIII. RNA Processing.
A. RNA Trimming.
B. RNA Splicing.
C. RNA Editing.
IX. Control of Translation.
X. Viral Maturation and Encapsidation.
XI. Nuclease in Defense Mechanism.
XII. Apoptosis and Nucleic Acid Salvage.
12. Nucleases and Human Diseases: Basis for Application.
I. Involvement of Nucleases in Human Disease.
A. Xeroderma Pigmentosum.
B. Ataxia Telangiectasia.
C. Cockayne Syndrome.
E. Aging–Werner Syndrome.
F. Immunological Diseases.
G. Nucleases and Neurological Disorders.
H. Human Diseases Involving Defective Protein Folding.
I. Other Human Diseases.
II. Reverse Genetics, Human Diseases, and Nucleases.
III. Use of Nucleases in Control of Human Disease.
13. Nucleases as Tools.
I. Nature of ′′Transforming Principle′′ as DNA.
II. Isolation of DNA and RNA.
III. Nearest–Neighborhood Analysis.
IV. Isolation of a Gene.
V. Uniparental Transmission During Cytoplasmic Inheritance.
VI. Physical Mapping of DNA.
VII. Use of Nuclease in the Development of Recombinant DNA Technology and the Molecular Cloning of a Gene.
VIII. Construction of an Artificial Chromosome.
IX. New Method for Mapping Eukaryotic Chromosomes.
A. Chromosome Walking (Overlap Hybridization).
B. Role of Nucleases in Transposon Mobility.
X. Use of Nuclease in the Physical Mapping of a Mutational Site.
XI. Biological Activity of a DNA Segment.
A. Use of Nucleases in the Identification of the Function of a DNA Segment via Transformation Experiments.
B. Use of Nucleases in the Deletion Mapping of Biological Activity.
C. Use of Nuclease in Identification of the Function of DNA Segment via Marker Rescue Method.
XII. Organization of Eukaryotic Chromosomes.
XIII. Distinction Between Active and Inactive Genes: The Relation Between Activity of a Gene and a Nuclease–Sensitive Site.
XIV. DNase Footprinting.
XV. Construction of Mutants: Site–Specific Mutation and Protein Engineering.
XVI. Nucleases in Directed Mutagenesis.
XVII. Nick Translation and Labeling of DNA with High–Specificity Radioactivity.
XVIII. Role of Nucleases in PCR.
A. Proofreading by Nuclease During PCR Amplification.
B. Application of 50 Nuclease in PCR Assay for Rapid Detection of a Known Gene in a DNA Sample(s).
C. Application of Nucleases in SNP–Genotyping and Pharmacogenetics.
XIX. Gene Knockout.
XX. RNase Protection Assay.
XXI. Use of Nucleases in Forensic Science.
XXII. Human and Other Genome Projects.
14. Application of Nucleases in Biotechnology, Medicine, Industry, and Environments.
I. Construction of Recombinant DNA and Molecular Cloning of Genes.
II. Biotechnology of Microorganisms, Plants, Animals and Marine Organisms Based on Recombinant DNA Technology.
III. Application in Medicine.
A. Role of Nucleases in Predictive, Preventive, and Curative Medicine.
B. Drug Designs.
C. Antisense Strategy.
IV. Nuclease Therapeutics and Therapeutic Targets.
A. DNaseI and DNAzyme–Based Therapeutics.
B. RNaseA and Ribozyme–Based Therapeutics.
C. Gene Therapy and Enhancement Therapy.
D. Gene Silencing.
E. RNaseL and Interferon–Mediated Control of Viral Infection and treatment of Cancer.
F. Recombinase–Mediated Control of Gene Expression.
G. Poisoning of Topoisomerase–DNA Intermediates.
V. Application in Forensics.
VI. Application in Industry: Production of Flavor Enhancer of Food and Beverage.
VII. Application in Environmental Problems.
B. Detection of Microbial Pathogens to Prevent Bioterrorism by 5′ Nuclease in PCR Assay.
15. Nucleases and Evolution.
I. Ribozyme as Evidence for the Early World of RNA.
II. Chemzyme, Ribozyme, and Proteinzyme.
III. The Role of Recombinase in Evolution.
A. Present–Day Selfish DNA–Possible Origin From Transposon.
IV. Nucleases and Control of DNA Transactions and Their Roles in Evolution.
V. Role of Nucleases in Directed Mutagenesis: Adaptive Mutation an SOS Response.
VI. Nucleases as Multifunctional Molecules.
VII. DNA Sequence Analysis, Crystal Structure, and Bioinformatics.
VIII. Possible Horizontal Transmission of Nuclease Gene and Intron.
IX. Conclusions and Our Future in the World of Nucleases.