Mechanisms of DNA Recombination and Genome Rearrangements: Methods to Study Homologous Recombination

1st Edition, Volume 600 - February 17, 2018
Editors: Maria Spies, Anna Malkova
Language: English
Hardback ISBN:
9 7 8 - 0 - 1 2 - 8 1 4 4 2 9 - 9
eBook ISBN:
9 7 8 - 0 - 1 2 - 8 1 4 4 3 0 - 5

Mechanisms of DNA Recombination and Genome Rearrangements: Methods to Study Homologous Recombination, Volume 600, the latest release in the Methods in Enzymology series, continue… Read more

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Mechanisms of DNA Recombination and Genome Rearrangements: Methods to Study Homologous Recombination, Volume 600, the latest release in the Methods in Enzymology series, continues the legacy of this premier serial with quality chapters authored by leaders in the field.

Homologous genetic recombination remains the most enigmatic process in DNA metabolism. The molecular machines of recombination preserve the integrity of the genetic material in all organisms and generate genetic diversity in evolution. The same molecular machines that support genetic integrity by orchestrating accurate repair of the most deleterious DNA lesions, however, also promote survival of cancerous cells and emergence of radiation and chemotherapy resistance. This two-volume set offers a comprehensive set of cutting edge methods to study various aspects of homologous recombination and cellular processes that utilize the enzymatic machinery of recombination The chapters are written by the leading researches and cover a broad range of topics from the basic molecular mechanisms of recombinational proteins and enzymes to emerging cellular techniques and drug discovery efforts.

1. Processing of DNA Double-Strand Breaks in Yeast
Robert Gnügge, Julyun Oh and Lorraine S. Symington

2. Methods to Study DNA End Resection I: Recombinant Protein Purification
Roopesh Anand, Cosimo Pinto and Petr Cejka

3. Methods to Study DNA End Resection II: Biochemical Reconstitution Assays
Cosimo Pinto, Roopesh Anand and Petr Cejka

4. Direct Quantitative Monitoring of Homology-Directed DNA Repair of Damaged Telomeres
Priyanka Verma, Robert L. Dilley, Melina T. Gyparaki and Roger A. Greenberg

5. Kinetic Analysis of the Exonuclease Activity of the Bacteriophage T4 Mre11–Rad50 Complex
Tibebe A. Teklemariam, Osvaldo D. Rivera and Scott W. Nelson

6. Observation and Analysis of RAD51 Nucleation Dynamics at Single-Monomer Resolution
Shyamal Subramanyam, Colin D. Kinz-Thompson, Ruben L. Gonzalez and Maria Spies

7. Determining the RAD51-DNA Nucleoprotein Filament Structure and Function By Cryo-Electron Microscopy
Lingyun Zhao, Jingfei Xu, Weixing Zhao, Patrick Sung, and Hong-Wei Wang

8. Expression, Purification, and Biochemical Evaluation of Human RAD51 Protein
Shyamal Subramanyam and Maria Spies

9. TIRF-Based Single-Molecule Detection of the RecA Presynaptic Filament Dynamics
Sung H. Kim

10. The RadA Recombinase and Paralogs of the Hyperthermophilic Archaeon Sulfolobus Solfataricus
Michael L. Rolfsmeier and Cynthia A. Haseltine

11. Reconstituting the 4-Strand DNA Strand Exchange
Olga M. Mazina and Alexander V. Mazin

12. Purification Of Saccharomyces Cerevisiae Homologous Recombination Proteins Dmc1 and Rdh54/Tid1 and a Fluorescent D-Loop Assay
Yuen-Ling Chan and Douglas K. Bishop

13. Probing Dynamic Assembly and Disassembly of Rad51 Tuned by Srs2 Using smFRET
Yupeng Qiu, Hye R. Koh and Sua Myong

14. The Recombination Mediator BRCA2: Architectural Plasticity of Recombination Intermediates Revealed by Single-Molecule Imaging (SFM/TIRF)
Arshdeep Sidhu, Dejan Ristic, Humberto Sánchez and Claire Wyman

15. Single-Molecule Dynamics and Localization of DNA Repair Proteins in Cells
Maarten W. Paul, Alex N. Zelensky, Claire Wyman and Roland Kanaar

16. Single-Stranded DNA Curtains for Studying the Srs2 Helicase Using Total Internal Reflection Fluorescence Microscopy
Luisina De Tullio, Kyle Kaniecki and Eric C. Greene

17. Single-Molecule Analysis of Replication Protein A–DNA Interactions
Fletcher E. Bain, Laura A. Fischer, Ran Chen and Marc S. Wold

18. Single-Molecule Studies of ssDNA-Binding Proteins Exchange
Olivia Yang and Taekjip Ha

19. Dissecting the Recombination Mediator Activity of BRCA2 Using Biochemical Methods
Catharina von Nicolai, Åsa Ehlén, Juan S. Martinez and Aura Carreira

20. Approaches to Understanding the Mediator Function of Brh2 in Ustilago maydis
Qingwen Zhou, William K. Holloman and Milorad Kojic

21. GEN1 Endonuclease: Purification and Nuclease Assays
Ying Wai Chan and Stephen C. West

22. Biochemical and Structural Properties of Fungal Holliday Junction-Resolving Enzymes
Yijin Liu, Alasdair Freeman, Anne-Cécile Déclais, Anton Gartner and David M. J. Lilley

23. Preparation and Resolution of Holliday Junction DNA Recombination Intermediates
Rajvee Shah Punatar and Stephen C. West

Maria Spies

Graduate of Peter the Great St. Petersburg Polytechnic University, Russia (1996 MS diploma with honors (equivalent of cum laude) in physics/biophysics) and Osaka University, Japan (2000 PhD in biological sciences), Dr. Maria Spies is an Associate Professor of Biochemistry at the University of Iowa Carver College of Medicine. Spies’ research career has been focused on deciphering the intricate choreography of the molecular machines orchestrating the central steps in the homology directed DNA repair. Her doctoral research supported by the Japanese Government (MONBUSHO) Graduate Scholarship provided the first detailed biochemical characterization of archaeal recombinase RadA. In her postdoctoral work with Dr. Steve Kowalczykowski (UC Davis) supported by the American Cancer Society, Spies reconstituted at the single-molecule level the initial steps of bacterial recombination and helped to explain how this process is regulated. Spies’ laboratory at the University of Iowa emphasizes the molecular machinery of homologous recombination, how it is integrated into DNA replication, repair and recombination (the 3Rs of genome stability), and how it is misappropriated in the molecular pathways that process stalled DNA replication events and DNA breaks through highly mutagenic, genome destabilizing mechanisms. Her goal is to understand, reconstitute and manipulate an elaborate network of DNA recombination, replication and repair, and to harness this understanding for anticancer drug discovery. The Spies lab utilizes a broad spectrum of techniques from biochemical reconstitutions of the key biochemical reactions in DNA recombination, repair and replication, to structural and single-molecule analyses of the proteins and enzymes coordinating these reactions, to combined HTS/CADD campaigns targeting human DNA repair proteins. Work in Spies Lab has been funded by the American Cancer Society (ACS), Howard Hughes Medical Institute (HHMI), and is currently supported by the National Institutes of Health (NIH). She received several prestigious awards including HHMI Early Career Scientist Award and Margaret Oakley Dayhoff Award in Biophysics. She serves on the editorial board of the Journal of Biological Chemistry, and as an academic editor of the journal Plos-ONE. She is a permanent member and a chair of the American Cancer Society “DNA mechanisms in cancer” review panel.

Affiliations and expertise

Carver College of Medicine, University of Iowa, USA

Anna Malkova

Graduate of the St. Petersburg State University, Russia (1987 MS diploma with honors (equivalent of cum laude) in Genetics and 1993 Ph.D. in Genetics), Dr. Anna Malkova is an Associate Professor of Biology Department at the University of Iowa College of Liberal Arts and Sciences. Malkova’s research is focused on DNA repair mechanisms. She investigates the repair of double-strand DNA breaks (DSBs), the most lethal type of DNA lesions. Using a dependable and powerful model system in yeast, where a single DSB is initiated by a site-specific HO endonuclease, she successfully demonstrated that imprecise or faulty repair of DSBs leads to structural genomic variations including mutations, copy number variations (CNVs), and chromosomal rearrangements similar to those that cause genetic diseases and cancer in humans. She has a longstanding interest in break-induced replication (BIR), a DSB repair pathway that repairs so-called one-ended DNA breaks, similar to those produced at eroded telomeres and by collapsed replication forks. In her postdoctoral work with Dr. James Haber (Brandeis University), she was among the first who identified this mechanism in eukaryotes. Studies in Malkova’s laboratory provided important insights into the mechanism of BIR, which was found to be fundamentally different from the mechanism of S-phase DNA replication. In particular, it was discovered that BIR is carried out by a migrating bubble rather than by a replication fork, and leads to conservative inheritance of newly synthesized DNA. This unusual molecular mechanism explained bursts of genetic instability that were observed in association with BIR, and that were similar to those associated with cancer and other diseases rooted in genetic instability. Work in Malkova’s lab is supported by the National Institutes of Health (NIH). Anna serves as a member to the Cancer Etiology study section of NIH/NCI.

Affiliations and expertise

Department of Biology, University of Iowa, Iowa City, Iowa, USA

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