Single Stranded DNA Binding Proteins (Methods in Molecular Biology, 2281)

Preface
Contents
Contributors
Chapter 1: The Essential, Ubiquitous Single-Stranded DNA-Binding Proteins
	1 The OB Fold and the General Mechanism of ssDNA Binding
	2 Classification of SSB Proteins
	3 Bacterial SSB Proteins
	4 Eukaryotic SSB Proteins
	5 The ``Other´´ Eukaryotic SSB Proteins
		5.1 Single-Stranded DNA-Binding Proteins 1 and 2
		5.2 Mitochondrial SSB Proteins
	6 Archaeal SSB Proteins
	7 Viral SSB Proteins
	References
Chapter 2: Single-Stranded DNA-Binding Proteins in the Archaea
	1 Introduction
	2 Phylogenetic Distribution of Archaeal SSB/RPA Proteins
	3 Euryarchaeal RPA Proteins
		3.1 RPA Proteins in Class I Methanogens
			3.1.1 Methanocaldococcus jannaschii
			3.1.2 Methanothermobacter thermautotrophicus
			3.1.3 Methanopyrus kandleri
		3.2 RPA Proteins in the Class II Methanogens
			3.2.1 Methanosarcina acetivorans
		3.3 RPA Proteins in the Halobacteriales
			3.3.1 Haloferax volcanii
			3.3.2 Halobacterium salinarum
		3.4 RPA Proteins in the Thermococcales
		3.5 RPA Proteins in the Thermoplasmatales
	4 Crenarchaeal SSB Proteins
		4.1 Sulfolobus solfataricus
		4.2 Displacement of SSB in the Thermoproteales
	5 Summary
	References
Chapter 3: Single-Molecule Fluorescence Methods to Study Protein Exchange Kinetics in Supramolecular Complexes
	1 Introduction
	2 Materials
		2.1 Glass-Surface Functionalization
		2.2 Microfluidic Flow Cell Device
		2.3 Exchange Assay Imaging
		2.4 Data Analysis
	3 Methods
		3.1 Chase Exchange Assay
			3.1.1 Glass-Surface Functionalization
			3.1.2 Constructing a Microfluidic Flow Cell Device
			3.1.3 Imaging the Chase Exchange Assay
			3.1.4 Data Quantification
		3.2 FRAP Exchange Assay
			3.2.1 Imaging the FRAP Exchange Assay
			3.2.2 Data Quantification
		3.3 Two-Color Exchange Assay
			3.3.1 Imaging the Two-Color Exchange Assay
			3.3.2 Data Quantification
	4 Notes
	References
Chapter 4: Comparing SSB-PriA Functional and Physical Interactions in Gram-Positive and -Negative Bacteria
	1 Introduction
	2 Materials
	3 Methods
		3.1 ConSurf Analysis
		3.2 SPR
			3.2.1 Preparation of Reagents and Samples
			3.2.2 Immobilization of SaPriA
			3.2.3 Binding of SaSsbA, SaSsbA-Ct, and KpSSB-Ct
			3.2.4 Analysis of the Sensorgrams
		3.3 ATPase Stimulation Assay
	4 Notes
	References
Chapter 5: In Vivo Binding of Single-Stranded DNA-Binding Protein to Stalled Replication Fork Helicases
	1 Introduction
	2 Materials
	3 Methods
		3.1 Dual/Triple-Plasmid Transformation
		3.2 Dual/Triple-Plasmid Co-expression and Cell Growth
		3.3 Protein Purification
			3.3.1 Purification of Histidine-Tagged Protein Using Nickel Column Chromatography
			3.3.2 Purification of Biotinylated Protein After Nickel Column Chromatography
			3.3.3 Purification of Profinity-Tagged Complexes
			3.3.4 Gel Filtration
		3.4 Fluorescence Microscopy
	4 Notes
	References
Chapter 6: Magnetic Tweezers-Based Single-Molecule Assays to Study Interaction of E. coli SSB with DNA and RecQ Helicase
	1 Introduction
	2 Materials
	3 Methods
		3.1 DNA Force-Extension Measurements to Study SSB Binding
		3.2 Rezipping Assays to Study SSB Binding
		3.3 Constant Force Assays to Explore the Different SSB Binding Modes
		3.4 Interaction Between SSB and RecQ Helicase
			3.4.1 Analyzing RecQ Variants
			3.4.2 Stimulation of RecQ Helicase Activity by  SSB
	4 Notes
	References
Chapter 7: High-Throughput Screening to Identify Inhibitors of SSB-Protein Interactions
	1 Introduction
	2 Materials
		2.1 Protein Expression
		2.2 Protein Purification
		2.3 AS Pilot and  AS
		2.4 AS Counterscreen
		2.5 FA Screening
	3 Methods
		3.1 Protein Expression
		3.2 Protein Purification
		3.3 AS Pilot Screen
		3.4 Selection of a Screening Library
		3.5 AS
		3.6 Data Analysis
		3.7 AS: Counterscreen (See Note 21)
		3.8 FA Screening
		3.9 Hit Validation
	4 Notes
	References
Chapter 8: Single-Molecule Tethered Particle Motion Studies on the DNA Recombinase Filament Assembly and Disassembly
	1 Introduction
	2 Materials
		2.1 Preparation of Dual-Labeled Gapped DNA Substrates
			2.1.1 Preparation of DNA Precursor with 5′ ssDNA Overhang Using Auto-Sticky PCR
			2.1.2 Preparation of 5′-Phosphorylated Poly dT Oligos via Enzymatic Phosphorylation
			2.1.3 Preparation of DNA Precursor with 3′ Poly dT ssDNA Overhang via DNA Ligation
			2.1.4 DNA Substrate Purification
		2.2 Preparation of Streptavidin-Coupled Polystyrene Beads
		2.3 Cleaning Glass Slides for the Reaction Chamber
		2.4 Glass Slide Passivation (Silanization)
		2.5 Preparation of Reaction Chamber
		2.6 Reaction Buffers
		2.7 Single-Molecule Tethered Particle Motion Assembly and Disassembly Experiments
	3 Methods
		3.1 Preparation of Dual-Labeled Gapped DNA Substrates
			3.1.1 Preparation of DNA Precursor with 5′ ssDNA Overhang Using Auto-Sticky PCR
			3.1.2 Preparation of 5′-Phosphorylated Poly dT Oligos via Enzymatic Phosphorylation
			3.1.3 Preparation of DNA Precursor with 3′ Poly dT ssDNA Overhang Via DNA Ligation
		3.2 Preparation of Streptavidin-Coupled Polystyrene Beads
		3.3 Cleaning Glass Slides for the Reaction Chamber
		3.4 Glass Slide Passivation (Silanization)
		3.5 Preparation of Reaction Chamber
		3.6 Single-Molecule Tethered Particle Motion Assembly Experiments
		3.7 Single-Molecule Tethered Particle Motion Disassembly Experiments
	4 Notes
	References
Chapter 9: Generation of Fluorescent Versions of Saccharomyces cerevisiae RPA to Study the Conformational Dynamics of Its ssDN...
	1 Introduction
	2 Materials
		2.1 Buffer Solutions
		2.2 Commercial Reagents
		2.3 Plasmids for RPA Overproduction and 4AZP Incorporation
	3 Methods
		3.1 Synthesis of 4AZP
			3.1.1 Starting Material Suspension and Apparatus Setup
			3.1.2 Dichloromethane (DCM) Extraction of Product from Aqueous Phase
			3.1.3 Aqueous Extraction of Product
			3.1.4 Decrease Product Solubility
			3.1.5 Product Recrystallization
			3.1.6 Drying of Crystalline Product
			3.1.7 Measure the Yield of Crystalline Product and Determine Dryness
		3.2 Overproduction and Purification of 4AZP-Incorporated ScRPA
			3.2.1 Bacterial Transformation
			3.2.2 Minimal Media for 4AZP Incorporation
			3.2.3 Overproduction of RPA with 4AZP
			3.2.4 Growth of the RPA Culture
			3.2.5 Purification of RPA4AZP
			3.2.6 Labeling of RPA4AZP with Click Chemistry-Based Fluorophores
			3.2.7 Measure the Efficiency of Labeling
			3.2.8 Testing the Activity of the Labeled RPA
	4 Notes
	References
Chapter 10: RPA-1 from Leishmania sp.: Recombinant Protein Expression and Purification, Molecular Modeling, and Molecular Dyna...
	1 Introduction
	2 Materials
		2.1 Heterologous Expression of Recombinant RPA-1 from L. amazonensis
		2.2 Expression and Purification of Recombinant L. amazonensis RPA-1
		2.3 Molecular Modeling and Molecular Dynamics Simulations of RPA-1 from L. amazonensis
	3 Methods
		3.1 Heterologous Expression of Recombinant RPA-1 from L. amazonensis
		3.2 Recombinant RPA-1 from L. amazonensis Purification
		3.3 Molecular Modeling and Molecular Dynamics Simulations of RPA-1 from L. amazonensis
	4 Notes
	References
Chapter 11: Single-Stranded DNA Curtains for Single-Molecule Visualization of Rad51-ssDNA Filament Dynamics
	1 Introduction
		1.1 Homologous Recombination
		1.2 Single-Molecule Studies of  HR
		1.3 ssDNA Curtains
	2 Materials
	3 Methods
		3.1 Lipid Passivation and ssDNA Attachment
		3.2 Labeling ssDNA Using Fluorescently Tagged RPA
		3.3 Measuring Rad51 Assembly and Disassembly Kinetics
		3.4 Data Analysis for Rad51-ssDNA Assembly and Disassembly Kinetics
		3.5 Rad51 Disruption by Srs2
		3.6 Disassembly and Cleaning of Slides
	4 Notes
	References
Chapter 12: Following Trypanosoma cruzi RPA-DNA Interaction Using Fluorescent In Situ Hybridization Coupled with Immunofluores...
	1 Introduction
	2 Materials
	3 Methods
		3.1 Slide Preparation
		3.2 Fixation and Permeabilization
		3.3 Immunofluorescence
		3.4 Fluorescence In Situ Hybridization
		3.5 Analyze the Slide
	4 Notes
	References
Chapter 13: Quantifying the Affinity of Trypanosoma cruzi RPA-1 to the Single-Stranded DNA Overhang of the Telomere Using Surf...
	1 Introduction
	2 Materials
		2.1 General Materials and Buffers
		2.2 Streptavidin Immobilization
		2.3 DNA Binding to Streptavidin
		2.4 Regeneration Scouting, Protein-DNA Binding, and Kinetics
	3 Methods
		3.1 Streptavidin Immobilization
		3.2 DNA Binding to Streptavidin
		3.3 Regeneration Scouting
		3.4 Protein-DNA Binding
		3.5 Kinetics
	4 Notes
	References
Chapter 14: Expression, Purification, and Solution-State NMR Analysis of the Two Human Single-Stranded DNA-Binding Proteins hS...
	1 Introduction
	2 Materials
		2.1 Preparation, Cloning, and Transformation of hSSB Constructs
		2.2 15N- and 13C-Labeled Recombinant hSSB Protein Expression
		2.3 hSSB Protein Purification
		2.4 NMR Spectroscopy
	3 Methods
		3.1 Preparation, Cloning, and Transformation of hSSB Constructs
			3.1.1 Transformation and Preparation of hSSB Constructs
			3.1.2 Cloning of Prepared hSSB Constructs into Expression Vector (pGEX-6P)
		3.2 15N- and 13C-Labeled Recombinant hSSB Protein Expression
			3.2.1 Expression Day 1
			3.2.2 Expression Day 2
			3.2.3 Single-Labeled 15N Expression
			3.2.4 Double-Labeled 15N-13C Expression
		3.3 hSSB Protein Purification
			3.3.1 Cell Lysis
			3.3.2 Glutathione Sepharose (GSH) Affinity Chromatography
			3.3.3 Heparin Affinity Chromatography
			3.3.4 Protein Gels (SDS-PAGE)
		3.4 NMR Spectroscopy
	4 Notes
	References
Chapter 15: Atomic Force Microscopy Reveals that the Drosophila Telomere-Capping Protein Verrocchio Is a Single-Stranded DNA-B...
	1 Introduction
	2 Materials
		2.1 Oligonucleotide Sequences
		2.2 Preparation of the DNA Substrates and Binding with the Protein
		2.3 Substrate Preparation and Deposition on the Mica
		2.4 AFM Imaging and Analysis
	3 Methods
		3.1 Preparation of the DNA Substrates
		3.2 Preparation of the Deposition Substrate for AFM Imaging
		3.3 DNA Substrate Deposition on the Mica
		3.4 DNA-Protein Complex Preparation and Deposition on the Mica Surface
		3.5 AFM Imaging in Tapping Mode
		3.6 Binding Position Analysis
		3.7 Analysis of the Stoichiometry of ssDNA-Binding Proteins
	4 Notes
	References
Chapter 16: Analysis of Mitochondrial SSB-DNA Complexes and Their Effects on DNA Polymerase γ Activity by Electron Microscopy ...
	1 Introduction
	2 Materials
		2.1 Sample Preparation for EM
		2.2 EM Analysis
		2.3 Assay of Stimulation of Pol γ Activity by Human mtSSB
	3 Methods
		3.1 Sample Preparation for EM
		3.2 EM Analysis
		3.3 Assay of Stimulation of Pol γ Activity by Human mtSSB
	4 Notes
	References
Chapter 17: Optical Tweezers to Investigate the Structure and Energetics of Single-Stranded DNA-Binding Protein-DNA Complexes
	1 Introduction
	2 Materials
		2.1 Polystyrene Bead Functionalization
		2.2 Fluidic Chambers
		2.3 Reaction Buffers
		2.4 Preparation of DNA Hairpins
	3 Methods
		3.1 Preparation of Fluidic Chambers for Optical Tweezers Studies
		3.2 Preparation of Functionalized Polystyrene Beads
		3.3 Preparation of DNA Hairpin
			3.3.1 Preparation of the Unwinding Segment
			3.3.2 Preparation of Digoxigenin-Labeled dsDNA Handles
			3.3.3 Preparation of dsDNA Spacer
			3.3.4 Preparation of a Linker DNA Segment
			3.3.5 Preparation of a DNA Loop for Hairpin Apex
			3.3.6 Preparation of Final DNA Hairpin Constructs
		3.4 Generation and Manipulation of Single ssDNA Molecules in the Optical Tweezers
			3.4.1 Incubation of the DNA Hairpin Construct with Anti-digoxigenin-Coated Beads
			3.4.2 Generation of Single-DNA Hairpin Tethers in the Optical Tweezers
			3.4.3 Identification of Individual DNA Hairpins
			3.4.4 Generation of ssDNA in the Optical Tweezers
		3.5 Mechanical Characterization of SSB-DNA Complexes
			3.5.1 Preparation of SSB Solution
			3.5.2 Determination of Force-Extension Curves of SSB-ssDNA Complexes
		3.6 Calculation of the Energy to Unwrap a Single Nucleotide from the SSB Tetramer
		3.7 Determination of the SSB-Binding Mode to ssDNA
		3.8 Determination of SSB-Binding Mode During DNA Replication
	4 Notes
	References
Chapter 18: Measurements of Real-Time Replication Kinetics of DNA Polymerases on ssDNA Templates Coated with Single-Stranded D...
	1 Introduction
	2 Materials
		2.1 Polystyrene Bead Functionalization
		2.2 Flow Chambers
		2.3 Hybrid Single-Double-Stranded DNA (ssdsDNA) Preparation
		2.4 Denaturing Alkaline Gel Electrophoresis
		2.5 Replication Reaction
	3 Methods
		3.1 Preparation of Fluidic Chambers for Optical Tweezers Studies
		3.2 Preparation of Functionalized Polystyrene Beads
		3.3 Preparation of Hybrid ssdsDNA Molecules for Manipulation with Optical Tweezers
			3.3.1 Production of ssdsDNA Molecules
			3.3.2 Preparation of Digoxigenin-Labeled dsDNA Handles (DIG-DNA Handle)
			3.3.3 Biotin-Labeled DNA Handle
			3.3.4 DNA Ligation
		3.4 Isolation and Manipulation of Single ssdsDNA Molecules in the Optical Tweezers
			3.4.1 DNA-Bead Incubation
			3.4.2 Isolation of Individual ssdsDNA Molecules in the Optical Tweezers
		3.5 Detection of Individual DNA Replication Activities on SSB-Free and SSB-Bound ssDNA
			3.5.1 Preparation of a Reaction Solution Containing the DNA Polymerase with/Without  SSB
			3.5.2 Measurement of Replication Activities on SSB-Free and SSB-Bound ssDNA
	4 Notes
	References
Chapter 19: Selective Suppression of Endogenous Gene Expression Using RNAi in Drosophila Schneider S2 Cells
	1 Introduction
	2 Materials
		2.1 RNAi in Schneider Cells
			2.1.1 Construction of Exogenous mtSSB Expression Vectors and the RNAi Vector
			2.1.2 Establishment of the Cell Lines
	3 Methods
		3.1 Selective Suppression of Endogenous Drosophila mtSSB in Schneider Cells
			3.1.1 Construction of the mtSSB Expression Vector
			3.1.2 Construction of the RNAi Vector
			3.1.3 Establishment of Cell Lines
		3.2 Induction of dsRNA Expression with or Without Exogenous mtSSB
		3.3 Mitochondrial DNA Analysis
	4 Notes
	References
Chapter 20: Stimulation of Variant Forms of the Mitochondrial DNA Helicase Twinkle by the Mitochondrial Single-Stranded DNA-Bi...
	1 Introduction
	2 Materials
	3 Methods
		3.1 dsDNA Unwinding Assay
		3.2 Helicase Stimulation Assay
	4 Notes
	References
Chapter 21: Measuring the Complex Effects of the Single-Stranded DNA-Binding Protein gp2.5 on Primer Synthesis and Extension b...
	1 Introduction
	2 Materials
		2.1 Effect of gp2.5 on Primer Synthesis
		2.2 Effect of gp2.5 on Primer Extension
		2.3 Denaturing Polyacrylamide Gel Electrophoresis
		2.4 Data Analysis
	3 Methods
		3.1 Effect of gp2.5 on Primer Synthesis
			3.1.1 Multiple-Turnover Primer Synthesis: Manual Sampling
			3.1.2 Multiple-Turnover Primer Synthesis Reaction: Rapid-Quench Instrument
			3.1.3 Single-Turnover Primer Synthesis Reaction: Rapid-Quench Instrument
		3.2 Effect of gp2.5 on Primer Extension
			3.2.1 Multiple-Turnover Primer Synthesis and Extension Reaction: Manual Sampling
			3.2.2 Multiple-Turnover Primer Synthesis and Extension Reaction: Rapid-Quench Instrument
			3.2.3 Single-Turnover Primer Synthesis and Extension Reaction Using a Rapid-Quench Instrument (See Note 10)
		3.3 Denaturing Polyacrylamide Gel Electrophoresis
		3.4 Data Analysis
	4 Notes
	References
Chapter 22: Strand Displacement and Unwinding Assays to Study the Concerted Action of the DNA Polymerase and SSB During Phi29 ...
	1 Introduction
	2 Materials
		2.1 Incubation Reaction
		2.2 8% Polyacrylamide Gels, 0.1% SDS (300 x 250 x 0.5 mm)
		2.3 Alkaline 0.7% Agarose  Gels
		2.4 Nucleotides and  DNAs
	3 Methods
		3.1 Strand Displacement Coupled to M13-DNA Replication
		3.2 TP-DNA Replication with an Exonuclease-Deficient Phi29 DNA Polymerase
		3.3 Unwinding Assay
	4 Notes
	References
Chapter 23: Structural Characterization of a Single-Stranded DNA-Binding Protein: A Case Study of the ORF6 Protein from Bacter...
	1 Introduction
	2 Materials
		2.1 Construct Design
		2.2 Protein Production
		2.3 Protein Purification
		2.4 Crystallization
		2.5 Software for Structure Determination and Analysis
	3 Methods
		3.1 Construct Design
		3.2 Protein Production
		3.3 Protein Purification
		3.4 Crystallization
		3.5 Data Collection
		3.6 Structure Determination
			3.6.1 Data Reduction
			3.6.2 Phasing
			3.6.3 Model Building, Refinement, and Validation
		3.7 Structural Analysis
	4 Notes
	References
Index