ME T H O D S I N MO L E C U L A R B I O L O G Y

Series EditorJohn M. Walker

School of Life and Medical SciencesUniversity of Hertfordshire

Hatfield, Hertfordshire, AL10 9AB, UK

For further volumes:http://www.springer.com/series/7651

DNA Repair

Methods and Protocols

Edited by

Lata Balakrishnan

Indiana University – Purdue University, Indianapolis, IN, USA

Jason A. Stewart

Department of Biological Sciences, University of South Carolina, Columbia, SC, USA

EditorsLata BalakrishnanIndiana University – Purdue UniversityIndianapolis, IN, USA

Jason A. StewartDepartment of Biological SciencesUniversity of South CarolinaColumbia, SC, USA

ISSN 1064-3745 ISSN 1940-6029 (electronic)Methods in Molecular BiologyISBN 978-1-4939-9499-1 ISBN 978-1-4939-9500-4 (eBook)https://doi.org/10.1007/978-1-4939-9500-4

© Springer Science+Business Media, LLC, part of Springer Nature 2019This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material isconcerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproductionon microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation,computer software, or by similar or dissimilar methodology now known or hereafter developed.The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply,even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulationsand therefore free for general use.The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed tobe true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty,express or implied, with respect to the material contained herein or for any errors or omissions that may have been made.The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Preface

Maintenance of genome integrity is important for all aspects of cellular physiology anddevelopment. Alterations in our genome due to endogenous and exogenous factors threatenthe integrity of our genome and therein have a direct consequence to human health.Evolution of DNA repair mechanisms has ensured that the majority of the DNA damageacquired is often quickly and efficiently reversed to restore genome stability. Based on thetype of damage encountered, different repair proteins are mobilized to the site of damage forDNA repair. Over the past five decades, our understanding of these repair pathways hasincreased by convergent knowledge from chemists, biochemists, geneticists, molecularbiologists, radiobiologists, and oncologists, among many others. At the heart of under-standing the molecular details of these complex processes is the wealth of information thathas been gained by the development of a wide range of experimental techniques.

This edition of Methods in Molecular Biology will outline techniques that will serve as aguide to assist researchers in their experimental design as they study the underlying mechan-isms of these important DNA repair pathways. The chapters in this book have been dividedinto five general parts, each focused on specific aspects of repair biology.

In Part I, two timely reviews are provided on different aspects of DNA repair. Chapter 1discusses the role of post-translational modifications on homology-directed repair (HDR)and recent advances in the treatment of HDR-defective cancers. The second review focuseson DNA damage caused by defective telomere replication, with a focus on telomere fragilityin cells utilizing the recombination-based alternative lengthening of telomeres (ALT)pathway.

Part II, titled “Cellular Assays to Detect and Measure DNA Damage and DamageResponse,” describes methods in the detection and measurement of DNA breaks andproteins involved in the DNA damage response (DDR). Chapter 3 describes in detail theuse of micro-irradiation to create DSBs followed by the use of live-cell imaging or immuno-fluorescence (IF) to quantify the dynamic association and dissociation of proteins involvedin both the recognition and repair of DSBs. In Chapter 4, Pond and Ellis provide a detaileddescription of the use of pulse-field gel electrophoresis (PFGE) to detect and measure DSBsin genomic DNA. Chapter 5 then shifts focus to the detection of tri-nucleotide repeat(TNR) expansions and deletions caused by DNA base lesions. In this method, Lai et al.describe how to measure the distribution and location of these lesions and their affect TNRstability. Finally, Chapter 6 presents a qPCR method to measure cellular nucleotide pools,which, when not properly maintained, leads to DNA damage and defective DNA repair.

Part III, titled “Cellular and Cell Extract-Based Assays to Measure DNA Repair,”encompasses methods designed to measure DNA repair efficiency and characterize factorsinvolved in the repair process. Firstly, Chapter 7 focuses on a much-needed method tomeasure HDR following genome-editing (i.e., CRISPR, TALEN) through a fluorescenceconversion assay, which allows for flow cytometry-based detection of converted cells fol-lowed by DNA sequencing to characterize the conversion tracts. Chapter 8 follows up withanother reporter-based assay to directly measure base-excision repair (BER) in human cells.The next two chapters make use of Xenopus egg extracts, as a cell-free method to detect

v

DNA repair. Chapter 9 describes a newly developed assay, which creates site-specific ssDNAbreaks on plasmid DNA. This plasmid can then be incubated with Xenopus egg extracts tomeasure ssDNA repair efficiency as well as protein recruitment and the DDR. Chapter 10makes use of Xenopus extracts to perform chromatin-immunoprecipitation (ChIP) to mea-sure dynamic protein associations and displacement in a synchronized, cell-free system,which has distinct advantages over cell-based assays. Chapter 11 then describes the use ofcell viability assays, IF, and metaphase spread analysis to understand the functional impor-tance of DNA helicases during DNA repair. The final two chapters of this part describemethods to screen for genetic complementation following DNA damage by the colonyformation assay (Chapter 12) and the identification of small molecules that inhibit DNArepair proteins (Chapter 13).

Part IV, titled “In Vitro Biochemical and Biophysical Methods to Study DNA Repair,”outlines different in vitro assays using purified recombinant proteins to study DNA repairenzyme mechanisms. Chapter 14 outlines the expression and purification of three proteinsinvolved in DSB repair and use of a single-molecule DNA curtain assay to temporally andspatially assess the mechanisms of DNA resection end resection at DSBs. In Chapter 15,Sausen et al. describe two techniques to study the ATPase activity of recombinant helicase/ATPase enzymes. The first outlines a direct method for assaying ATPase activity usingradiolabeled ATP and thin layer chromatography and the second uses a high-throughput96-well plate assay that couples ATPase activity with nicotinamide adenine dinucleotide(NADH) oxidation for real-time monitoring. Chapter 16 details biochemical methods toassess the efficiency of repair enzymes during different DNA transactions, namely, DNAunwinding and annealing. Strand exchange mechanisms are frequently observed duringhomologous recombination and repair and Chapters 16 and 17 outline methodologies tostudy strand exchange reactions with purified recombination proteins. With a similar focuson the HDR pathway, Chapters 18 and 19 describe methodologies to assess branchmigration and nucleoprotein filament formation, respectively.

The tail end of the book focuses on methods to study DNA damage and repair withinthe telomeric regions and/or arising during DNA replication. Part V is titled “Methods toAssess Telomeric and Replication-Induced DNA Damage and Repair.” Fouquerel et al. inChapter 20 present highly sensitive protocols, which allow for measurement of UV photo-products that induce telomere damage in human cells and subsequent assessment of theirremoval and repair. Genome stability is directly impacted by the dynamics of replication forkmovement during the duplication process. DNA fiber analysis allows for direct assessment offork movement and replication dynamics. Assays describing DNA fiber methods to measuregenome-wide and telomere replication are presented in Chapters 21 and 22, respectively.The presence of guanine-rich sequences at telomere ends promote the formation ofG-quadraplex structures, which both protect the ends of the chromosomes as well as impederepair and replication. Chapter 23 outlines an in vivo method to detect these structures andtheir colocalization to telomeric regions using IF. The final technique (Chapter 24)describes the use of FISH to detect DNA damage on metaphase chromosomes, usingtelomeric FISH probes.

Many of the techniques outlined in this book can be easily adapted or modified byresearchers interested in studying DNA transactions to assess their specific protein or repairpathway of interest. Our hope is that these methods will be useful for both those new andwell-established in the field and help advance our understanding of DNA repair. We would

vi Preface

like to specifically acknowledge and thank all the authors who contributed to this book withtheir expert descriptions and detailed outlines of various protocols, which will undoubtedlybe an excellent resource to many in the field. We would also like to thank the series editor,John Walker, for assistance in the development and editing of this book.

Indianapolis, IN, USA Lata BalakrishnanColumbia, SC, USA Jason A. Stewart

Preface vii

Contents

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vContributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiii

PART I REVIEWS

1 Homologous Recombination-Mediated DNA Repair and Implicationsfor Clinical Treatment of Repair Defective Cancers. . . . . . . . . . . . . . . . . . . . . . . . . . 3Nicole M. Reilly, Brian D. Yard, and Douglas L. Pittman

2 Resolving Roadblocks to Telomere Replication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31Emily Mason-Osann, Himabindu Gali, and Rachel Litman Flynn

PART II CELLULAR ASSAYS TO DETECT AND MEASURE DNADAMAGE AND DAMAGE RESPONSE

3 In Time and Space: Laser Microirradiation and the DNADamage Response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61Jae Jin Kim, Ramhari Kumbhar, Fade Gong, and Kyle M. Miller

4 Quantification of Double-Strand Breaks in Mammalian CellsUsing Pulsed-Field Gel Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75Kelvin W. Pond and Nathan A. Ellis

5 Methods to Study Trinucleotide Repeat Instability Inducedby DNA Damage and Repair . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87Yanhao Lai, Ruipeng Lei, Yaou Ren, and Yuan Liu

6 Extracting and Measuring dNTP Pools in Saccharomyces cerevisiae . . . . . . . . . . . . 103Radha Subramaniam, Natalie A. Lamb, Yoonchan Hwang,Lauren Johengen, and Jennifer A. Surtees

PART III CELLULAR AND CELL EXTRACT-BASED ASSAYS TO MEASURE

DNA REPAIR

7 Conversion Tract Analysis of Homology-Directed GenomeEditing Using Oligonucleotide Donors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131Yinan Kan and Eric A. Hendrickson

8 Reporter Assays for BER Pathway. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145Dorota Piekna-Przybylska

9 Methods for Studying DNA Single-Strand Break Repair and Signalingin Xenopus laevis Egg Extracts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161Yunfeng Lin, Anh Ha, and Shan Yan

ix

10 Chromatin Immunoprecipitation (ChIP) of Plasmid-Bound Proteinsin Xenopus Egg Extracts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173Kelly B. Wolfe and David T. Long

11 Cellular Assays to Study the Functional Importance of HumanDNA Repair Helicases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185Sanket Awate, Srijita Dhar, Joshua A. Sommers, and Robert M. Brosh Jr.

12 A Mammalian Genetic Complementation Assay for Assessing CellularResistance to Genotoxic Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209Nicole M. Reilly and Douglas L. Pittman

13 Small-Molecule Inhibitor Screen for DNA Repair Proteins. . . . . . . . . . . . . . . . . . . 217John J. Turchi and Pamela S. VanderVere-Carozza

PART IV IN VITRO BIOCHEMICAL AND BIOPHYSICAL METHODS

TO STUDY DNA REPAIR

14 Assembling the Human Resectosome on DNA Curtains. . . . . . . . . . . . . . . . . . . . . 225Michael M. Soniat, Logan R. Myler, and Ilya J. Finkelstein

15 Thin-Layer Chromatography and Real-Time Coupled Assaysto Measure ATP Hydrolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245Christopher W. Sausen, Cody M. Rogers, and Matthew L. Bochman

16 Gel-Based Assays for Measuring DNA Unwinding, Annealing,and Strand Exchange . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255Cody M. Rogers, Christopher W. Sausen, and Matthew L. Bochman

17 In Vitro Assay for Plasmid Length DNA Strand Exchangeby Human DMC1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265Steven D. Goodson, Russell B. Hawes, Sarah M. Waldvogel,and Michael G. Sehorn

18 In Vitro Assays for DNA Branch Migration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271Andrew A. Kelso, Steven D. Goodson, and Michael G. Sehorn

19 Stabilization of the Human DMC1 Nucleoprotein Filament . . . . . . . . . . . . . . . . . 285Sarah M. Waldvogel, Steven D. Goodson, and Michael G. Sehorn

PART V METHODS TO ASSESS TELOMERIC AND REPLICATION-INDUCED

DNA DAMAGE AND REPAIR

20 Measuring UV Photoproduct Repair in Isolated Telomeresand Bulk Genomic DNA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295Elise Fouquerel, Ryan P. Barnes, Hong Wang, and Patricia L. Opresko

21 Single-Molecule DNA Fiber Analyses to Characterize ReplicationFork Dynamics in Living Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 307Srijita Dhar, Arindam Datta, Taraswi Banerjee, and Robert M. Brosh Jr.

22 Direct Visualization of DNA Replication at Telomeres UsingDNA Fiber Combing Combined with Telomere FISH . . . . . . . . . . . . . . . . . . . . . . 319Himabindu Gali, Emily Mason-Osann, and Rachel Litman Flynn

x Contents

23 Telomere and G-Quadruplex Colocalization Analysisby Immunofluorescence Fluorescence In Situ Hybridization (IF-FISH) . . . . . . . 327Miaomiao Zhang, Rui Liu, and Feng Wang

24 FISHing for Damage on Metaphase Chromosomes . . . . . . . . . . . . . . . . . . . . . . . . . 335P. Logan Schuck and Jason A. Stewart

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 349

Contents xi

Contributors

SANKET AWATE � Laboratory of Molecular Gerontology, National Institute on Aging, NIH,NIH Biomedical Research Center, Baltimore, MD, USA

TARASWI BANERJEE � DSFederal, Rockville, MD, USARYAN P. BARNES � Department of Environmental and Occupational Health, UPMC

Hillman Cancer Center, University of Pittsburgh, Pittsburgh, PA, USAMATTHEW L. BOCHMAN � Department of Molecular and Cellular Biochemistry, Indiana

University, Bloomington, IN, USAROBERT M. BROSH JR � Laboratory of Molecular Gerontology, National Institute on Aging,

NIH, NIH Biomedical Research Center, Baltimore, MD, USAARINDAM DATTA � Laboratory of Molecular Gerontology, National Institute on Aging, NIH,

NIH Biomedical Research Center, Baltimore, MD, USASRIJITA DHAR � Laboratory of Molecular Gerontology, National Institute on Aging, NIH,

NIH Biomedical Research Center, Baltimore, MD, USANATHAN A. ELLIS � Department of Cellular and Molecular Medicine, University of Arizona

Cancer Center, University of Arizona, Tuscon, AZ, USAILYA J. FINKELSTEIN � Department of Molecular Biosciences and Institute for Cellular and

Molecular Biology, The University of Texas at Austin, Austin, TX, USA; Center for Systemsand Synthetic Biology, The University of Texas at Austin, Austin, TX, USA

RACHEL LITMAN FLYNN � Department of Pharmacology and Experimental Therapeutics,Boston University School of Medicine, Boston, MA, USA; Department of Medicine, CancerCenter, Boston University School of Medicine, Boston, MA, USA

ELISE FOUQUEREL � Department of Biochemistry and Molecular Biology, Thomas JeffersonUniversity, Philadelphia, PA, USA; Department of Environmental and OccupationalHealth, UPMC Hillman Cancer Center, University of Pittsburgh, Pittsburgh, PA, USA

HIMABINDU GALI � Department of Pharmacology and Experimental Therapeutics, BostonUniversity School of Medicine, Boston, MA, USA; Department of Medicine, Cancer Center,Boston University School of Medicine, Boston, MA, USA

FADE GONG � Department of Molecular Biosciences, Institute for Cellular and MolecularBiology, The University of Texas at Austin, Austin, TX, USA; Department of Biochemistryand Molecular Biology, Baylor College of Medicine, Houston, TX, USA

STEVEN D. GOODSON � Department of Genetics and Biochemistry, Clemson University,Clemson, SC, USA; Eukaryotic Pathogens Innovation Center, Clemson University,Clemson, SC, USA

ANH HA � Department of Biological Sciences, University of North Carolina at Charlotte,Charlotte, NC, USA

RUSSELL B. HAWES � Department of Genetics and Biochemistry, Clemson University,Clemson, SC, USA

ERIC A. HENDRICKSON � BMBB Department, University of Minnesota Medical School,Minneapolis, MN, USA

YOONCHAN HWANG � Department of Biochemistry, Jacobs School of Medicine and BiomedicalSciences, State University of New York at Buffalo, Buffalo, NY, USA

LAUREN JOHENGEN � Department of Biochemistry, Jacobs School of Medicine and BiomedicalSciences, State University of New York at Buffalo, Buffalo, NY, USA

xiii

YINAN KAN � eGenesis, Cambridge, MA, USAANDREWA. KELSO � Department of Genetics and Biochemistry, Clemson University, Clemson,

SC, USA; Eukaryotic Pathogens Innovation Center, Clemson University, Clemson, SC,USA

JAE JIN KIM � Department of Molecular Biosciences, Institute for Cellular and MolecularBiology, The University of Texas at Austin, Austin, TX, USA

RAMHARI KUMBHAR � Department of Molecular Biosciences, Institute for Cellular andMolecular Biology, The University of Texas at Austin, Austin, TX, USA

YANHAO LAI � Department of Chemistry and Biochemistry, Florida International University,Miami, FL, USA

NATALIE A. LAMB � Department of Biochemistry, Jacobs School of Medicine and BiomedicalSciences, State University of New York at Buffalo, Buffalo, NY, USA

RUIPENG LEI � Biochemistry Ph.D. Program, Florida International University, Miami, FL,USA

YUNFENG LIN � Department of Biological Sciences, University of North Carolina atCharlotte, Charlotte, NC, USA

YUAN LIU � Department of Chemistry and Biochemistry, Florida International University,Miami, FL, USA; Biochemistry Ph.D. Program, Florida International University, Miami,FL, USA; Biomolecular Sciences Institute, Florida International University, Miami, FL,USA

RUI LIU � Department of Genetics, School of Basic Medical Sciences, Tianjin MedicalUniversity, Tianjin, People’s Republic of China

DAVID T. LONG � Department of Biochemistry and Molecular Biology, Medical University ofSouth Carolina, Charleston, SC, USA

EMILY MASON-OSANN � Department of Pharmacology and Experimental Therapeutics,Boston University School of Medicine, Boston, MA, USA; Department of Medicine, CancerCenter, Boston University School of Medicine, Boston, MA, USA

KYLE M. MILLER � Department of Molecular Biosciences, Institute for Cellular andMolecular Biology, The University of Texas at Austin, Austin, TX, USA

LOGAN R. MYLER � Department of Molecular Biosciences and Institute for Cellular andMolecular Biology, The University of Texas at Austin, Austin, TX, USA

PATRICIA L. OPRESKO � Department of Environmental and Occupational Health, UPMCHillman Cancer Center, University of Pittsburgh, Pittsburgh, PA, USA

DOROTA PIEKNA-PRZYBYLSKA � Department of Microbiology and Immunology, School ofMedicine and Dentistry, University of Rochester, Rochester, NY, USA

DOUGLAS L. PITTMAN � Department of Drug Discovery and Biomedical Sciences, College ofPharmacy, University of South Carolina, Columbia, SC, USA

KELVIN W. POND � Department of Cellular and Molecular Medicine, University of ArizonaCancer Center, University of Arizona, Tuscon, AZ, USA

NICOLE M. REILLY � Fondazione Piemontese per la Ricerca sul Cancro ONLUS, Candiolo,Italy

YAOU REN � Biochemistry Ph.D. Program, Florida International University, Miami, FL,USA

CODY M. ROGERS � Department of Molecular and Cellular Biochemistry, IndianaUniversity, Bloomington, IN, USA

CHRISTOPHER W. SAUSEN � Department of Molecular and Cellular Biochemistry, IndianaUniversity, Bloomington, IN, USA

xiv Contributors

P. LOGAN SCHUCK � Department of Biological Sciences, University of South Carolina,Columbia, SC, USA

MICHAEL G. SEHORN � Department of Genetics and Biochemistry, Clemson University,Clemson, SC, USA; Eukaryotic Pathogens Innovation Center, Clemson University,Clemson, SC, USA; Clemson University School of Health Research, Clemson, SC, USA;Center for Optical Materials Science and Engineering Technologies, Clemson University,Clemson, SC, USA

JOSHUA A. SOMMERS � Laboratory of Molecular Gerontology, National Institute on Aging,NIH, NIH Biomedical Research Center, Baltimore, MD, USA

MICHAEL M. SONIAT � Department of Molecular Biosciences and Institute for Cellular andMolecular Biology, The University of Texas at Austin, Austin, TX, USA

JASON A. STEWART � Department of Biological Sciences, University of South Carolina,Columbia, SC, USA

RADHA SUBRAMANIAM � Genetics, Genomics and Bioinformatics Program, Jacobs School ofMedicine and Biomedical Sciences, State University of New York at Buffalo, Buffalo, NY,USA

JENNIFER A. SURTEES � Genetics, Genomics and Bioinformatics Program, Jacobs School ofMedicine and Biomedical Sciences, State University of New York at Buffalo, Buffalo, NY,USA; Department of Biochemistry, Jacobs School of Medicine and Biomedical Sciences, StateUniversity of New York at Buffalo, Buffalo, NY, USA

JOHN J. TURCHI � Department of Medicine, Indiana University School of Medicine,Indianapolis, IN, USA; Department of Biochemistry and Molecular Biology, IndianaUniversity School of Medicine, Indianapolis, IN, USA

PAMELA S. VANDERVERE-CAROZZA � Department of Medicine, Indiana University School ofMedicine, Indianapolis, IN, USA

SARAH M. WALDVOGEL � Department of Genetics and Biochemistry, Clemson University,Clemson, SC, USA

HONG WANG � Department of Physics, North Carolina State University, Raleigh, NC, USA;Center for Human Health and the Environment, North Carolina State University,Raleigh, NC, USA

FENG WANG � Department of Genetics, School of Basic Medical Sciences, Tianjin MedicalUniversity, Tianjin, People’s Republic of China

KELLY B. WOLFE � Department of Biochemistry and Molecular Biology, Medical University ofSouth Carolina, Charleston, SC, USA

SHAN YAN � Department of Biological Sciences, University of North Carolina at Charlotte,Charlotte, NC, USA

BRIAN D. YARD � Department of Translational Hematology and Oncology Research, LernerResearch Institute, Cleveland Clinic, Cleveland, OH, USA

MIAOMIAO ZHANG � Department of Genetics, School of Basic Medical Sciences, TianjinMedical University, Tianjin, People’s Republic of China

Contributors xv