Summary
The detailed understanding of the DNA replication process requires structural insight. The combination of psoralen crosslinking and electron microscopy has been extensively exploited to reveal the fine architecture of in vivo DNA replication intermediates. This approach proved instrumental to uncover the basic mechanisms of DNA duplication, as well as the perturbation of this process by genotoxic treatments. The replication structures need to the stabilized in vivo (by psoralen crosslinking) prior to extraction and enrichment procedures, finally leading to the visualization at the transmission electron microscope. This chapter outlines the procedures required to visualize in vivo replication intermediates of genomic DNA, extracted from budding yeast or cultured mammalian cells.
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
Inciarte, M. R., Salas, M., and Sogo, J. M. (1980) Structure of replicating DNA molecules of Bacillus subtilis bacteriophage phi 29. Journal of Virology 34, 187–99.
Lucchini, R., and Sogo, J. M. (1995) Replication of transcriptionally active chromatin. Nature 374, 276–80.
Sogo, J. M., Stahl, H., Koller, T., and Knippers, R. (1986) Structure of replicating simian virus 40 minichromosomes. The replication fork, core histone segregation and terminal structures. Journal of Molecular Biology 189, 189–204.
Avemann, K., Knippers, R., Koller, T., and Sogo, J. M. (1988) Camptothecin, a specific inhibitor of type I DNA topoisomerase, induces DNA breakage at replication forks. Molecular and Cellular Biology 8, 3026–34.
Lopes, M., Foiani, M., and Sogo, J. M. (2006) Multiple mechanisms control chromosome integrity after replication fork uncoupling and restart at irreparable UV lesions. Molecular Cell 21, 15–27.
Mojas, N., Lopes, M., and Jiricny, J. (2007) Mismatch repair-dependent processing of methylation damage gives rise to persistent single-stranded gaps in newly replicated DNA. Genes and Development 21, 3342–55.
Sogo, J. M., Lopes, M., and Foiani, M. (2002) Fork reversal and ssDNA accumulation at stalled replication forks owing to checkpoint defects [see comment]. Science 297, 599–602.
Gasser, R., Koller, T., and Sogo, J. M. (1996) The stability of nucleosomes at the replication fork. Journal of Molecular Biology 258, 224–39.
Gruss, C., Wu, J., Koller, T., and Sogo, J. M. (1993) Disruption of the nucleosomes at the replication fork. EMBO J 12, 4533–45.
Wellinger, R. E., Lucchini, R., Dammann, R., and Sogo, J. M. (1999) In vivo mapping of nucleosomes using psoralen-DNA crosslinking and primer extension. Methods in Molecular Biology 119, 161–73.
Gruber, M., Wellinger, R. E., and Sogo, J. M. (2000) Architecture of the replication fork stalled at the 3’′ end of yeast ribosomal genes. Molecular Cellular Biology 20, 5777–87.
Vollenweider, H. J., Sogo, J. M., and Koller, T. (1975) A routine method for protein-free spreading of double- and single-stranded nucleic acid molecules. Proceedings of the National Academy of Sciences of the United States of America 72, 83–7.
Sogo, J. M., and Thoma, F. (1989) Electron microscopy of chromatin. Methods in Enzymology 170, 142–65.
Acknowledgments
I am wholeheartedly grateful to Dr. José M. Sogo, not only for his precious advice compiling this manuscript, but also for the patient coaching while learning this technique and the continuous support to my scientific career. I wish to thank the whole team at the EMEZ (Electron Microscopy Center of the ETH Zurich) and at the ZMB (Center for Microscopy and Image Analysis of the University Zurich) for excellent technical assistance running the EM experiments. Special thanks go to Dr. Andres Kaech, whose assistance has been precious for the recent establishment of this technology at the University of Zurich. I am also grateful to Dr. Diana Santelia for her help with the pictures and to Dr. Kai Neelsen for critical reading of the manuscript. The work in my lab is financed by Swiss National Science Foundation grant n. PP00A--114922, associated to my SNF professorship.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Lopes, M. (2009). Electron Microscopy Methods for Studying In Vivo DNA Replication Intermediates. In: Vengrova, S., Dalgaard, J. (eds) DNA Replication. Methods in Molecular Biology, vol 521. Humana Press. https://doi.org/10.1007/978-1-60327-815-7_34
Download citation
DOI: https://doi.org/10.1007/978-1-60327-815-7_34
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-60327-814-0
Online ISBN: 978-1-60327-815-7
eBook Packages: Springer Protocols