Main description:

Ataxia-telangiectasia (A-T) is a rare and severe genetic disorder affecting children. A-T is a multisystem disease characterized by progressive neurodegeneration, immunodeficiency and cancer predisposition. This detailed volume explores the ever expanding field of research into the ATM (ataxia-telangiectasia, mutated) gene and the role played by ATM kinase in DNA damage signaling and diverse cellular processes. What follows is a handy desktop reference for both seasoned A-T researchers and postgraduate students, as it demonstrates the breadth of recent developments in A-T studies. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Both classic and cutting-edge techniques are described, including ATM gene mutation detection, assays for radiosensitivity and radioresistant DNA synthesis, new methods to measure ATM kinase activity by imaging microscopy and high content screening as well as proteomics, phosphoproteomics and bioinformatics approaches to decipher ATM-dependent signalling pathways. Additional methods include generation of patient-specific stem cells and approaches to study ATM functions in the nervous system.
Comprehensive and practical, ATM Kinase: Methods and Protocols aims to ignite and attract the interest of colleagues from diverse fields to A-T research in an effort to bring their expertise and fresh ideas to resolve many A-T puzzles still waiting to be pieced together and to alleviate the suffering of A-T children and their families.

Contents:

1. Assaying Radiosensitivity of Ataxia-Telangiectasia

Hailiang Hu, Shareef Nahas, and Richard A. Gatti

2. Assaying for Radioresistant DNA Synthesis, the Hallmark Feature of the Intra-S-Phase Checkpoint Using a DNA Fiber Fluorography-Based Methodology

Amanda W. Kijas and Martin F. Lavin

3. ATM Gene Mutation Detection Techniques and Functional Analysis

Guillaume Rieunier, Catherine Dubois D'Enghien, Alice Fievet, Dorine Bellanger, Dominique Stoppa-Lyonnet, and Marc-Henri Stern

4. An HTRF (R) Assay for the Protein Kinase ATM

Phillip Adams, Jonathan Clark, Simon Hawdon, Jennifer Hill, and Andrew Plater

5. ATM Kinase Inhibitors: HTS Cellular Imaging Assay Using CellomicsTM ArrayScan VTI Platform

Catherine Bardelle and Joanna Boros

6. Image-Based High Content Screening: Automating the Quantification Process for DNA Damage-Induced Foci

Yi Chieh Lim

7. Analyzing ATM Function by Electroporation of Endonucleases and Immunofluorescence Microscopy

Keiji Suzuki

8. Quantitative and Dynamic Imaging of ATM Kinase Activity by Bioluminescence Imaging

Shyam Nyati, Grant Young, Brian Dale Ross, and Alnawaz Rehemtulla

9. Zn(II)-Phos-Tag SDS-PAGE for Separation and Detection of a DNA Damage-Related Signaling Large Phosphoprotein

Eiji Kinoshita, Emiko Kinoshita-Kikuta, and Tohru Koike

10. Identification of ATM Protein Kinase Phosphorylation Sites by Mass Spectrometry

Mark E. Graham, Martin F. Lavin, and Sergei V. Kozlov

11. Studies of ATM Kinase Activity Using Engineered ATM Sensitive to ATP Analogues (ATM-AS)

Masato Enari, Yuko Matsushima-Hibiya, Makoto Miyazaki, and Ryo Otomo

12. Functional Characterization of ATM Kinase Using Acetylation-Specific Antibodies

Sun Yingli and Du Fengxia

13. Identification of ATM-Interacting Proteins by Co-Immunoprecipitation and Glutathione-S-Transferase (GST) Pull-Down Assays

Amanda L. Bain, Janelle L. Harris, and Kum Kum Khanna

14. ATM Activation and H2AX Phosphorylation Induced by Genotoxic Agents Assessed by Flow- and Laser Scanning-Cytometry

Hong Zhao, H. Dorota Halicka, Jorge Garcia, Jiangwei Li, and Zbigniew Darzynkiewicz

15. Peptide Immunoaffinity Enrichment with Targeted Mass Spectrometry: Application to Quantification of ATM Kinase Phospho-Signaling

Jeffrey R. Whiteaker, Lei Zhao, Regine M. Schoenherr, Jacob J. Kennedy, Richard G. Ivey, and Amanda G. Paulovich

16. Mass Spectrometry-Based Proteomics for Quantifying DNA Damage-Induced Phosphorylation

Marina E. Borisova, Sebastian A. Wagner, and Petra Beli

17. Statistical Analysis of ATM-Dependent Signaling in Quantitative Mass Spectrometry Phosphoproteomics

Ashley J. Waardenberg

18. ChIP Technique to Study Protein Dynamics at Defined DNA Double-Strand Breaks

Jie Wen and Patrick Concannon

19. Studies of the DNA Damage Response by Using the Lac Operator/Repressor (LacO/LacR) Tethering System

Rossana Piccinno, Marta Cipinska, and Vassilis Roukos

20. Imaging of Fluorescently-Tagged ATM Kinase at the Sites of DNA Double-Strand Breaks

Anthony J. Davis, Shih-Ya Wang, David J. Chen, and Benjamin P.C. Chen

21. Live Cell Imaging to Study Real-Time ATM-Mediated Recruitment of DNA Repair Complexes to Sites of Ionizing Radiation-Induced DNA Damage

Burkhard Jakob and Gisela Taucher Scholz

22. Analyzing Heterochromatic DNA Double Strand Break (DSB) Repair in Response to Ionizing Radiation

Karolin Klement and Aaron A. Goodarzi

23. Phenotypic Analysis of ATM Protein Kinase in DNA Double-Strand Break Formation and Repair

Elisabeth Mian and Lisa Wiesmuller

24. Monitoring DNA Repair Consequences of ATM Signaling Using Simultaneous Fluorescent Readouts

Adrian Wiegmans

25. Non-Canonical ATM Activation and Signaling in Response to Transcription-Blocking DNA Damage

Jurgen A. Marteijn, Wim Vermeulen, and Maria Tresini

26. Study of ATM Phosphorylation by Cdk5 in Neuronal Cells

Hua She and Zixu Mao

27. DNA Damage Response in Human Stem Cells and Neural Descendants

Jason M. Beckta, Bret R. Adams, and Kristoffer Valerie

28. A Patient-Specific Stem Cell Model to Investigate the Neurological Phenotype Observed in Ataxia-Telangiectasia

Romal Stewart, Gautam Wali, Chris Perry, Martin Lavin, Francois Feron, Alan Mackay-Sim, and Ratneswary Sutharsan

29. Lentiviral Reprogramming of A-T Patient Fibroblasts to Induced Pluripotent Stem Cells

Sam Nayler, Sergei Kozlov, Martin Lavin, and Ernst Wolvetang

30. Monitoring the ATM-Mediated DNA Damage Response in the Cerebellum Using Organotypic Cultures

Efrat Tal and Yosef Shiloh