R-Loops: Methods and Protocols

ISBN-13: 9781071624760 / Angielski / Twarda / 2022

Aguilera, Andrés

Opis

This detailed book compiles a series of laboratory protocols covering the most important aspects of R-loop biology. Beginning with a range of methods allowing for the detection of DNA-RNA hybrids, as well as their purification and visualization by electron microscopy, the volume continues with methods based on the use of RNase H-derived tools to detect DNA-RNA hybrids in vitro and in vivo. Several protocols permit studying non-canonical RNA nucleotides in the R-loop context, as well as a number of specific protocols devoted to the investigation of R-loop topology and their functional roles in the biology of mitochondria and telomeres. Finally, a large block of chapters is dedicated to different methods allowing genome-wide mapping of DNA-RNA hybrids in various organisms. 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. 

Authoritative and practical, R-Loops: Methods and Protocols serves as an ideal resource for those working on R-loop homeostasis but also to scientists studying such areas of molecular and cell biology as genome integrity, DNA replication and repair, chromatin remodeling, transcription, RNA processing, modification and export, as well as for researchers elucidating the molecular mechanisms of cancer and genetic diseases.

Spis treści

1. Direct R-Loop Visualization on Genomic DNA by Native Automated Electron Microscopy

            Henriette Stoy, Joel Luethi, Fabienne Roessler, Johannes Riemann, Andres Kaech, and Massimo Lopes

 

2. Detection of R-Loop Structures by Immunofluorescence Using the S9.6 Monoclonal Antibody

            Konstantina Skourti-Stathaki

 

3. R-Loop Detection in Bacteria

            Émilie Vlachos-Breton and Marc Drolet

 

4. Detection of R-Loops by In Vivo and In Vitro Cytosine Deamination in Saccharomyces cerevisiae

            Juan C. Cañas, Andrés Aguilera, and Belén Gómez-González

 

5. Identification of RNA-DNA Hybrids Associated with R-Loops at the IgH Switch Sequence in Activated B Cells

            Junghyun Lim, Brice Laffleur, Uttiya Basu, and Kefei Yu

 

6. Locus-Specific Analysis of Replication Dynamics and Detection of DNA:RNA Hybrids by Immuno Electron Microscopy

            Henriette Stoy, Kevin Lang, Houra Merrikh, and Massimo Lopes

 

7. RNase H1, the Gold Standard for R-Loop Detection

            Susana M. Cerritelli, Kiran Sakhuja, and Robert J. Crouch

 

8. RNase H1 Hybrid-Binding Domain-Based Tools for Cellular Biology Studies of DNA-RNA Hybrids in Mammalian Cells

            Sónia Silva, Cristina Guillén-Mendoza, and Andrés Aguilera

 

9. Detection and Quantification of RNA Modifications on RNA/DNA Hybrids Using SID–UPLC–MS/MS

            Marta Starczak, Abdulkadir Abakir, Alexey Ruzov, and Daniel Gackowski

 

10. DNA:RNA Hybrids at Telomeres in Budding Yeast

            Carolin B. Wagner and Brian Luke

 

11. Detection of TERRA R-Loops at Human Telomeres

            Galina Glousker, Rita Valador Fernandes, Marianna Feretzaki, and Joachim Lingner

 

12. R-Loops and Mitochondrial DNA Metabolism

            Ian J. Holt

 

13. Highly-Purified Top1-Bound DNA Fragments

            Jessica Marinello and Giovanni Capranico

 

14. R-Loop Immunoprecipitation: A Method to Detect R-Loop Interacting Factors In Vivo

            Chiara Beghè and Natalia Gromak

 

15. Studies on Protein–RNA:DNA Hybrid Interactions by Microscale Thermophoresis (MST)

            Miaomiao Li, Arne Klungland, and Bjørn Dalhus

 

16. Studying R-Loop Recognizing Proteins Using Single-Molecule DNA Curtain Technique and Electrophoretic Mobility Shift Assay

            Na Young Cheon and Ja Yil Lee

 

17. Immunostaining and Protein Pull-Down of Methyl-5-Cytosine-Marked RNA:DNA Hybrids

            Haibo Yang and Li Lan

 

18. In Vitro Binding of GADD45A to RNA:DNA Hybrids

            Khelifa Arab and Christof Niehrs

 

19. Proximity Ligation Assay for Detection of R-Loop Complexes upon DNA Damage

            Adele Alagia, Ruth F. Ketley, and Monika Gullerova

 

20. Biochemical Analysis of RNA-DNA Hybrid and R-Loop Unwinding via Motor Proteins

            Arijit Dutta, Youngho Kwon, and Patrick Sung

 

21. Topology of RNA:DNA Hybrids and R-Loops in Yeast

            Yathish Jagadheesh Achar and Marco Foiani

 

22. Detecting and Mapping N6-Methyladenosine on RNA/DNA Hybrids

            Abdulkadir Abakir, Fahad Alenezi, and Alexey Ruzov

 

23. Genome-Wide Native R-Loop Profiling by R-Loop Cleavage Under Targets and Tagmentation (R-Loop CUT&Tag)

            Honghong Wang, Conghui Li, and Kaiwei Liang

 

24. Mapping R-Loops Using Catalytically Inactive RNaseH1 (R-ChIP)

            Xuan Zhang, Yajing Hao, and Xiang-Dong Fu

 

25. Targeted Nuclease Approaches for Mapping Native R-Loops

            Phillip Wulfridge, Qingqing Yan, and Kavitha Sarma

 

26. Quantitative DNA-RNA Immunoprecipitation-Sequencing with Spike-Ins

            Magdalena P. Crossley and Karlene A. Cimprich

 

27. DNA:RNA Immunoprecipitation from S. pombe Cells for qPCR and Genome-Wide Sequencing

            Laetitia Vachez, Camille Teste, and Vincent Vanoosthuyse

 

28. Genome-Wide Analysis of DNA-RNA Hybrids in Yeast by DRIPc-Seq and DRIP-Seq

            María García-Rubio, María E. Soler-Oliva, and Andrés Aguilera

 

29. Quantitative, Convenient, and Efficient Genome-Wide R-Loop Profiling by ssDRIP-Seq in Multiple Organisms

            Wei Xu, Kuan Li, Qin Li, Shuai Li, Jincong Zhou, and Qianwen Sun