Abstract
DNA replication is a fundamental process of life. Any perturbation of this process by endogenous or exogenous factors impacts on genomic stability and thereby on carcinogenesis. More recently, the replication machinery has been discovered as an interesting target for cancer therapeutic strategies. Given its high biological and clinical relevance, technologies for the analysis of DNA replication have attracted major attention. The so-called DNA fiber spreading technique is a powerful tool to directly monitor various aspects of the replication process by sequential incorporation of halogenated nucleotide analogs which later can be fluorescently stained and analyzed. This chapter outlines the use of the DNA fiber spreading technique for the analysis of replication dynamics and replication structures.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Aguilera & García-Muse (2013) Causes of genome instability. Annu Rev Genet 47:1–32
Sarni D, Kerem B (2017) Oncogene-induced replication stress drives genome instability and tumorigenesis. Int J Mol Sci 18(7):1339
Bournique E, DallÒsto M, Hoffmann JS, Bergoglio V (2018) Role of specialized DNA polymerases in the limitation of replicative stress and DNA damage transmission. Mutat Res 808:62–73
Flach J, Milyavsky M (2018) Replication stress in hematopoietic stem cells in mouse and man. Mutat Res 808:74–82
Gaillard H, Garcia-Muse T, Aguilera A (2015) Replication stress and cancer. Nat Rev Cancer 15(5):276–289
Hampp S, Kiessling T, Buechle K, Mansilla SF, Thomale J, Rall M, Ahn J, Pospiech H, Gottifredi V, Wiesmüller L (2016) DNA damage tolerance pathway involving DNA polymerase ι and the tumor suppressor p53 regulates DNA replication fork progression. Proc Natl Acad Sci U S A 113(30):4311–4319
Jackson DA, Pombo A (1998) Replicon clusters are stable units of chromosome structure: evidence that nuclear organization contributes to the efficient activation and propagation of s phase in human cells. J Cell Biol 140(6):1285–1295
Nieminuszczy J, Schwab RA, Niedzwiedz W (2016) The DNA fibre technique – tracking helicases at work. Methods 108:92–98
Gratzner HG (1982) Monoclonal antibody to 5-bromo and 5-iododeoxyuridine: a new reagent for detection of DNA replication. Science 218:474–475
Gray JW (1985) Monoclonal antibodies against bromodeoxyuridine (special issue). Cytometry 6:501–673
Aten JA, Bakker PJ, Stap J, Boschman GA, Veenhof CH (1992) DNA double labeling with IdUrd and CldUrd for spatial and temporal analysis of cell proliferation and DNA replication. Histochem J 25(5):251–259
Bugler B, Schmitt E, Aressy B, Ducommun B (2010) Unscheduled expression of CDC25B in S-phase leads to replicative stress and DNA damage. Mol Cancer 4:9–29
Tècher H, Koundrioukoff S, Azar D, Wilhelm T, Carignon S, Brison O, Debatisse M, Le Tallec B (2013) Replication dynamics: biases and robustness of DNA fiber analysis. J Mol Biol 425:4845–4855
Schlacher K, Christ N, Siaud N, Egashira A, Wu H, Jasin M (2011) Double-strand break repair-independent role for BRCA2 in blocking stalled replication fork degradation by MRE11. Cell 145(4):529–542
Acknowledgments
We cordially thank Dr. Vanesa Gottifredi as well as Dr. Sabrina F. Mansilla for teaching Stephanie Biber the DNA fiber spreading assay during an internship at the Cell Cycle and Genomic Stability Laboratory, Fundación Instituto Leloir-IIBBA, CONICET Buenos Aires, Argentina financed by the DFG Graduate School for Molecular Medicine at Ulm University (IGradU), during her PhD fellowship funded by the IGradU. We also thank Dr. Helmut Pospiech (University of Oulu, Finland and Leibnitz Institute on Aging, Jena, Germany) for general advice regarding the DNA fiber spreading assay.
This work was supported by the German Research Foundation (DFG, PA3 in Research Training Group 1789 “Cellular and Molecular Mechanisms in Aging,” CEMMA) and by the German Cancer Aid (Deutsche Krebshilfe #70112504).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2021 The Author(s)
About this protocol
Cite this protocol
Biber, S., Wiesmüller, L. (2021). Analysis of Replication Dynamics Using the Single-Molecule DNA Fiber Spreading Assay. In: Manfredi, J.J. (eds) Cell Cycle Checkpoints. Methods in Molecular Biology, vol 2267. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1217-0_4
Download citation
DOI: https://doi.org/10.1007/978-1-0716-1217-0_4
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-1216-3
Online ISBN: 978-1-0716-1217-0
eBook Packages: Springer Protocols