دانلود کتاب DNA Topoisomerases: Methods and Protocols (Methods in Molecular Biology, Vol. 582)

فهرست مطالب :

METHODS IN MOLECULAR BIOLOGY
DNA Topoisomerases
Preface
Contents
Contributors
Introduction: Emerging Themes in DNA Topoisomerase Research
1. The Topological States of DNA
2. DNA Topoisomerase Enzyme Activity
3. Cellular Functions of DNA Topoisomerases
References
Analysis of DNA Topoisomers, Knots, and Catenanes by Agarose Gel Electrophoresis
1. Introduction
1.1. Gel Electrophoretic Analysis of Linking-Number Topoisomers
1.1.1. One-Dimensional Agarose Gel Analysis of Topoisomers
1.1.2. Two-Dimensional Agarose Gel Analysis of Topoisomers
1.2. Gel Electrophoretic Analysis of DNA Knots and Catenanes
2. Materials
2.1. Plasmid DNA
2.2. Recombination Proteins and Topoisomerases
2.3. Solutions ( see Note 2)
3. Methods
3.1. Plasmid Purification
3.2. Preparation of Lk-Topoisomer Ladders
3.3. Preparation of Torus-Knot and Torus-Catenane Ladders by Phage-lambda Integrative Recombination
3.4. Preparation of Twist-Knot Ladders Using Type-II Topoisomerases (see Note 6)
3.5. One-Dimensional Gel Electrophoresis (see Note 7)
3.6. Two-Dimensional Gel Electrophoresis of Lk Topoisomers
3.7. Gel Analysis and Quantitation
4. Notes
References
Two-Dimensional Agarose Gel Electrophoresis of DNA Topoisomers
1. Introduction
1.1. Separation of Supercoiled Topoisomers by 2-D Electrophoresis
1.2. DNA Structural Conversion During 2-D Electrophoresis
2. Materials
2.1. Plasmid DNA
2.2. DNA Topoisomerase Protein
2.3. Electrophoresis Apparatus
2.4. Electrophoresis Solutions
3. Methods
3.1. Preparation of S. cerevisiae Plasmid DNA from Spheroplasts
3.2. Preparation of S. cerevisiae Plasmid DNA by Cell Disruption with Glass Beads
3.3. Generation of Topoisomers of Desired Linking Numbers
3.4. Electrophoresis
4. Notes
References
Cleavage of Plasmid DNA by Eukaryotic Topoisomerase II
1. Introduction
2. Materials
2.1. Assay Components
2.2. Gel Electrophoresis
3. Methods
4. Notes
References
Assays for the Preferential Binding of Human Topoisomerase I to Supercoiled DNA
1. Introduction
2. Materials
2.1. Topoisomerase I Protein
2.2. Gel Shift Assay
2.3. Filter Binding Assay
3. Methods
3.1. Preparation of Nicked Circular DNA for Both Assays
3.2. Gel Shift Assay
3.2.1. Preparation of Linear Plasmid DNA
3.2.2. Sample Preparation
3.2.3. Agarose Gel Electrophoresis
3.3. Filter Binding Assay
3.3.1. Sample Preparation
3.3.2. Filtration and Determination of Bound Radioactivity
4. Notes
References
Monitoring the Topoisomerase II DNA Gate Conformational Change with Fluorescence Resonance Energy Transfer
1. Introduction
2. Materials
2.1. Oligonucleotides
2.2. Polyacrylamide Gel Electrophoresis (PAGE) of Oligonucleotides
2.3. NAP-5 Gel Filtration of Oligonucleotides
2.4. Fluorescence Spectroscopy
3. Methods
3.1. Annealing Oligonucleotides
3.2. PAGE Purification of Oligonucleotides
3.3. NAP-5 Gel Filtration of Annealed Oligonucleotides
3.4. Fluorescence Spectroscopy of DNA Substrate
3.5. Processing of Fluorescence Spectra to Determine FRET Efficiency
4. Notes
References
Single-Molecule Magnetic Tweezers Studies of Type IB Topoisomerases
1. Introduction
1.1. Topoisomerase Activity
1.2. Magnetic Tweezers and DNA Manipulation
1.3. The Magnetic Tweezers Microscope
1.4. The Magnetic Tweezers Software
2. Materials
2.1. Magnetic Tweezers Microscope
2.2. Magnetic Tweezers Software
2.3. Flow Cells for Magnetic Tweezers
2.4. Buffer Solutions
2.5. DNA Constructs for Magnetic Tweezers
2.6. Superparamagnetic Beads
2.7. Topoisomerase Stocks
3. Methods
3.1. Assembly of Flow Cells for Magnetic Tweezers
3.2. Preparation of the Magnetic Tweezers DNA Constructs
3.2.1. Construction of the Supercos1-Lambda1,2 Vector
3.2.2. Construction of the DNA Construct for Labeling
3.2.3. Preparation and Ligation of the Biotin and Digoxigenin Handles
3.3. Preparation of Magnetic Beads and DNA for Magnetic Tweezers Measurements
3.3.1. Assembly of Tethered DNA Constructs in the Flow Cell
3.3.2. Calibration of the Tethered DNA in the Magnetic Tweezers
3.4. Monitoring DNA-Topoisomerase IB Interactions
3.5. Analysis of Topoisomerase IB Time Traces
4. Notes
References
Dissolution of Double Holliday Junctions by the Concerted Action of BLM and Topoisomerase IIIalpha
1. Introduction
2. Materials
2.1. Substrate Preparation
2.2. Dissolution Reaction
2.3. Electrophoresis and Detection
3. Methods
3.1. Preparation of Substrates
3.1.1. Labelling the Oligonucleotides
3.1.2. Annealing the Oligonucleotides
3.1.3. Ligation of the Oligonucleotides
3.1.4. Casting the Denaturing Gel
3.1.5. Gel Purification of the Substrate
3.2. Double Holliday Junction Dissolution
3.2.1. Enzyme Reaction
3.2.2. Electrophoresis and Detection
4. Notes
References
ChIP-on-Chip Analysis of DNA Topoisomerases
1. Introduction
2. Materials
2.1. Preparation of Magnetic Beads
2.2. Preparation of Chromatin Extracts and Immunoprecipitation
2.3. Washing the Beads and Crosslink Reversal
2.4. DNA Purification
2.5. DNA Amplification
2.6. DNAse Digestion
2.7. DNA Labeling
2.8. Hybridization
2.9. Washing, Staining, and Scanning of the Chips
3. Methods
3.1. Preparation of the Magnetic Beads
3.2. Preparation of Chromatin Extracts and Immunoprecipitation
3.3. Washing the Beads and Crosslink Reversal
3.4. DNA Purification
3.5. DNA Amplification
3.5.1. Method I
3.5.2. Method II
3.6. DNAse Digestion (see Note 16)
3.7. DNA Labeling
3.8. Hybridization
3.9. Washing, Staining, and Scanning of Chips
4. Notes
References
Analyzing Top2 Distribution on Yeast Chromosomes by Chromatin Immunoprecipitation
1. Introduction
2. Materials
2.1. Analysis of Top2 Association with Chromatin
2.2. Cell Culture and Lysis
2.3. Chromatin Immunoprecipitation
2.4. Semi-quantitative PCR Analysis of Recovered Chromatin
3. Methods
3.1. Cell Culture and Protein Preparation
3.2. Chromatin Immunoprecipitation
3.3. Semi-quantitative PCR Analysis of Immunoprecipitated Chromatin
4. Notes
References
Binding of DNA Topoisomerases I and II to Replication Origins
1. Introduction
2. Materials
2.1. Cell Culture
2.2. Topoisomerase I Mapping
2.3. Topoisomerase II Mapping
3. Methods
3.1. Cell Cultures
3.2. Topoisomerase I Mapping
3.3. Topoisomerase II Mapping
4. Notes
References
Cytometric Assessment of DNA Damage Induced by DNA Topoisomerase Inhibitors
1. Introduction
2. Materials
2.1. Reagents
2.2. Instrumentation
3. Methods
4. Notes
References
Analysis of the Topoisomerase II-Dependent Decatenation G2 Checkpoint and Checkpoint Kinases in Human Cells
1. Introduction
2. Materials
2.1. Human Cell Culture
2.2. Checkpoint Biology
2.3. Checkpoint Kinase Biochemistry
3. Methods
3.1. Assay of Decatenation G2 Checkpoint
3.1.1. Assaying G2/M Progression in Unsynchronized Cells
3.1.2. Assaying G2/M Progression in Synchronized Cells Treated with Colcemid
3.1.3. Assaying G2/M Progression in Unsynchronized Cells Treated with Colcemid
3.2. Quantification of Mitotic Cells by Flow Cytometry
3.3. Assay of MPF Kinase
3.3.1. Extraction and Isolation of Cyclin B1/Cdk1 Complexes
3.3.2. Assay of MPF Kinase Activity
3.3.3. Assay of Plk-1 Kinase Activity
4. Notes
References
Assaying Topoisomerase II Checkpoints in Yeast
1. Introduction
1.1. Cell Cycle Synchronization of Yeast Cells
1.2. Analysis of Spindle Morphologies in Yeast Cells
1.3. Analysis of Chromosome Arm Condensation in Yeast Cells
2. Materials
2.1. Yeast Strains and Plasmids
2.2. Yeast Cell Growth and Synchronization
2.3. FACScan Analysis
3. Methods
3.1. Cell Culture and Synchronization
3.2. Processing the Samples for Analysis by Fluorescence Microscopy
3.2.1. Analysis of Spindle Morphologies
3.2.2. Analysis of Chromosome Condensation
3.3. Fluorescence-Activated Cell Scanning (FACScan) Analysis of DNA Content
4. Notes
References
Cytological Analysis of Chromosome Structural Defects that Result from Topoisomerase II Dysfunction
1. Introduction
1.1. Protocols
1.2. Mitotic Chromosome Preparations
1.3. Impregnation of Silver into Chromosomes
2. Materials
2.1. Cell Culture and Synchrony
2.2. Chromosome Preparations
2.3. Silver Impregnation
3. Methods
3.1. Growing and Synchronizing the Cells
3.2. Preparation of Mitotic Chromosome Morphologies that Maintain the 3D-Organization of Chromosomes on the Mitotic Spindle
3.3. Impregnation of Silver into Fixed Mitotic Chromosomes
4. Notes
References
Top2 SUMO Conjugation in Yeast Cell Lysates
1. Introduction
2. Materials
2.1. Analysis of Top2 SUMO Conjugates
2.2. Cell Culture and Lysis
2.3. SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
2.4. Western Blot for SUMO Modified Top2
3. Methods
3.1. Cell Culture and Protein Preparation
3.2. SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
3.3. Transfer to Nitrocellulose and Western Blot for SUMO-Modified Top2
4. Notes
References
Analysis of SUMOylation of Topoisomerase IIalpha with Xenopus Egg Extracts
1. Introduction
2. Materials
2.1. Preparation of CSF Extracts from Xenopus Eggs
2.2. Preparation of Demembraned Sperm Nuclei
2.3. Chromosome Assembly and Isolation
2.4. Analysis of SUMOylation of Topoisomerase II
3. Methods
3.1. Preparation of CSF Extracts from Xenopus Eggs
3.2. Preparation of Demembraned Sperm Nuclei
3.3. Assembly and Isolation of Chromosomes or Interphase Chromatin
3.4. Analysis of SUMOylation of Topoisomerase II
4. Notes
References
The Dynamics of DNA Topoisomerase IIalpha in Living Cells
1. Introduction
2. Materials
2.1. Construction of the EGFP-Topo IIalpha Plasmid
2.2. Transfection of LLC-Pk Cells and Selection of Stable Line
2.3. Imaging and Photomanipulation
3. Methods
3.1. Construction of the EGFP-Topo IIalpha Plasmid
3.1.1. Construction of the His-Tagged Topo IIalpha Plasmid
3.1.2. Construction of EGFP-Tagged Topo IIalpha
3.2. Transfection of LLC-Pk Cells and Selection of a Stable Cell Line
3.3. Imaging and Photomanipulation
3.4. Data Analysis
4. Notes
References
Depletion and Mutation of Topoisomerase II in Animal Cells
1. Introduction
2. Stable Topo II Depletion/Mutation by Gene Targeting
3. Stable Topo II Depletion by shRNA Expression
4. Transient Topo II Depletion by siRNA Delivery
5. Isolation and Characterisation of Topo II Inhibitor-Resistant Cells
6. Related Methods
7. Summary and Conclusions
References
Subject Index