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Helicase Enzymes Part A
- 1st Edition, Volume 672 - August 5, 2022
- Editor: Michael Trakselis
- Language: English
- Hardback ISBN:9 7 8 - 0 - 3 2 3 - 9 1 4 7 6 - 5
- eBook ISBN:9 7 8 - 0 - 3 2 3 - 9 1 4 7 7 - 2
Methods in Enzymology serial highlights new advances in the field with this new volume presenting interesting chapters. Each chapter is written by an international board of au… Read more
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Request a sales quoteMethods in Enzymology serial highlights new advances in the field with this new volume presenting interesting chapters. Each chapter is written by an international board of authors.
- Provides the authority and expertise of leading contributors from an international board of authors
- Presents the latest release in Methods in Enzymology serials
- Updated release includes the latest information on Helicase Enzymes
Biochemists, biophysicists, molecular biologists, analytical chemists, and physiologists
- Cover image
- Title Page
- Table of Contents
- Copyright
- Contributors
- Preface
- Chapter One: Monitoring helicase-catalyzed unwinding of multiple duplexes simultaneously
- Abstract
- 1: Introduction
- 2: Key resource tables
- 3: Safety considerations and standards
- 4: Materials and equipment
- 5: Preparation of dual duplex DNA substrates
- 6: Measuring helicase-catalyzed unwinding of dual-duplex DNA substrates
- 7: Alternative methods/procedures
- References
- Chapter Two: Alignment of helicases on single-stranded DNA increases activity
- Abstract
- 1: Introduction
- 2: Preparation of an artificial dimeric helicase, NS3h-fusion
- 3: Characterization of the NS3h-fushion by DNA binding
- 4: DNA unwinding using the rapid quench-flow instrument from Kintek Corp
- 5: DNA footprinting to compare DNA binding of NS3H-FUSION to NS3h and NS3 full length helicase
- 6: Streptavidin displacement assay indicates NS3h-fusion can increase dissociation of streptavidin from a biotin-labeled oligonucleotide
- 7: Conclusions and summary
- Acknowledgments
- References
- Chapter Three: Use of an unnatural amino acid to map helicase/DNA interfaces via photoactivated crosslinking
- Abstract
- 1: Introduction
- 2: Design of Bpa incorporation sites
- 3: Expression/incorporation of Bpa into PriA
- 4: Design/construction of DNA replication forks
- 5: Determining strand-specific PriA-Bpa crosslinking
- 6: Primer extension to determine interaction points
- 7: Summary
- References
- Chapter Four: Quantitative methods to study helicase, DNA polymerase, and exonuclease coupling during DNA replication
- Abstract
- 1: Introduction
- 2: Real-time quantitation of unwinding-synthesis by coupled helicase-DNA polymerase
- 3: Aminopurine-based method to study cooperative base-pair melting at replication fork junction
- 4: Simultaneous measurement of polymerase and exonuclease activities using minicircle fork DNA
- Acknowledgments
- References
- Chapter Five: Assembling bacteriophage T7 leading-strand replisome for structural investigation
- Abstract
- 1: Introduction
- 2: Major considerations
- 3: Experimental protocols
- 4: Cryo-EM data analysis
- 5: Summary
- Acknowledgments
- References
- Chapter Six: In vivo fluorescent TUNEL detection of single stranded DNA gaps and breaks induced by dnaB helicase mutants in Escherichia coli
- Abstract
- 1: Introduction
- 2: Materials and equipment
- 3: Culture preparation
- 4: TUNEL labeling
- 5: Analyzing your TUNEL samples
- 6: Summary
- References
- Chapter Seven: Biochemical methods to monitor loading and activation of hexameric helicases
- Abstract
- 1: Introduction
- 2: Hexameric helicase loading assay
- 3: Fluorescence-based fork unwinding assay
- 4: Summary and discussion
- Acknowledgments
- References
- Chapter Eight: DNA fiber analyses to study functional importance of helicases and associated factors during replication stress
- Abstract
- 1: Introduction
- 2: DNA fiber assay using fiber spreading technique
- 3: Expected outcomes
- 4: Notes
- 5: Advantages
- 6: Limitations
- 7: Conclusions
- Acknowledgments
- References
- Chapter Nine: Optimizing CMG helicase and CMG-dependent replication assays by designing DNA fork substrates and choosing nucleotide analogues for helicase preloading
- Abstract
- 1: Introduction
- 2: Designing DNA fork substrates for studying CMG structure and function
- 3: Protocols for DNA fork preparation
- 4: Protocols for CMG helicase assays
- 5: Protocols for CMG-dependent replication assays
- 6: Summary
- Acknowledgments
- References
- Chapter Ten: ReconSil: An electron microscopy toolbox to study helicase function at an origin of replication
- Abstract
- 1: Introduction
- 2: Protein expressions and purifications
- 3: DNA template, OCCM complex preparation and staining
- 4: Image processing
- 5: Results
- 6: Summary
- Acknowledgments
- References
- Chapter Eleven: CMG helicase activity on G4-containing DNA templates
- Abstract
- 1: Introduction
- 2: CMG purification
- 3: DNA substrates
- 4: Helicase assay
- 5: Summary and conclusions
- Acknowledgment
- References
- Chapter Twelve: Detecting G4 unwinding
- Abstract
- 1: Introduction
- 2: Before you begin
- 3: Materials and equipment
- 4: Step-by-step method details
- Acknowledgments
- References
- Chapter Thirteen: Helicase mediated vectorial folding of telomere G-quadruplex
- Abstract
- 1: Introduction
- 2: Key resources table
- 3: Experimental preparation for vectorial folding
- 4: Helicase induced vectorial folding (vf)
- 5: Accessibility test
- 6: Conclusion
- Acknowledgments
- References
- Chapter Fourteen: Production of long linear DNA substrates with site-specific chemical lesions for single-molecule replisome studies
- Abstract
- 1: Introduction
- 2: Materials and methods
- 3: Validation of methods and controls
- 4: Summary and discussion
- References
- Chapter Fifteen: Replication fork collapse in vitro using Xenopus egg extracts
- Abstract
- 1: Introduction
- 2: Purification of lac repressor
- 3: Purification of tet repressor
- 4: Purification of nicked DNA
- 5: Analysis of nick stability in xenopus egg extracts
- 6: Replication fork collapse in xenopus egg extracts
- 7: Summary and conclusions
- Acknowledgments
- References
- Chapter Sixteen: Measuring the contributions of helicases to break-induced replication
- Abstract
- 1: Introduction
- 2: General method to grow yeast cells and induce DNA double-strand break
- 3: Analysis of protein contribution to BIR and its regulation in yeast cells
- 4: Analysis of genome instability in mutants deficient in extensive BIR synthesis
- 5: Analysis of DNA synthesis during BIR by ddPCR
- 6: Summary and conclusions
- Acknowledgments
- References
- Chapter Seventeen: A RADAR method to measure DNA topoisomerase covalent complexes
- Abstract
- 1: Introduction
- 2: Before you begin
- 3: Key resources table
- 4: Materials and equipment
- 5: Step-by-step method details
- 6: Expected outcomes
- 7: Quantification and statistical analysis
- 8: Advantages
- 9: Limitations
- 10: Optimization and troubleshooting
- Acknowledgments
- Reference
- No. of pages: 400
- Language: English
- Edition: 1
- Volume: 672
- Published: August 5, 2022
- Imprint: Academic Press
- Hardback ISBN: 9780323914765
- eBook ISBN: 9780323914772
MT
Michael Trakselis
Michael grew up in a western suburb of Chicago called Aurora, IL, or better known as Wayne’s World. He got his BS in Psychology and Chemistry at the University of Illinois, Urbana-Champaign. After which, he employed as a technician working on analytical method development at Abbott Laboratories. From 1998-2002 he was a PhD student at Penn State University working in Stephen Benkovic lab on the Bacteriophage T4 replisome assembly and dynamics. In 2003, he started a postdoctoral fellowship in Ron Laskey’s laboratory at the University of Cambridge/Hutchison MRC in the UK. In 2006, he started his independent career at the University of Pittsburgh Chemistry department. Starting 2014, he moved his laboratory to Baylor University, where he currently lives with his family. There, the Trakselis laboratory is currently studying aspects of DNA replication and repair spanning from the very in vitro biochemical to the very in vivo cell and human biology.
Affiliations and expertise
Associate Professor and Director of Graduate Affairs, Baylor University, TX, USARead Helicase Enzymes Part A on ScienceDirect