Visualization and interpretation of eukaryotic DNA replication intermediates in vivo by electron microscopy

The detailed understanding of the DNA replication process requires structural insight. The combination of psoralen cross-linking and electron microscopy has been extensively exploited to reveal the fine architecture of in vivo DNA replication intermediates. This approach proved instrumental to uncov...

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Bibliographic Details
Published in:Methods in molecular biology (Clifton, N.J.) Vol. 1094; p. 177
Main Authors: Neelsen, Kai J, Chaudhuri, Arnab Ray, Follonier, Cindy, Herrador, Raquel, Lopes, Massimo
Format: Journal Article
Language:English
Published: United States 01.01.2014
Subjects:
Animals
Cell Extracts
Chromatin - metabolism
Chromosomes, Artificial, Bacterial - metabolism
Cross-Linking Reagents - pharmacology
DNA - metabolism
DNA Replication - drug effects
DNA, Cruciform - metabolism
Eukaryotic Cells - cytology
Eukaryotic Cells - ultrastructure
Ficusin - pharmacology
Genome, Fungal
Male
Mammals
Microscopy, Electron - methods
Nucleic Acid Denaturation - drug effects
Saccharomyces cerevisiae - drug effects
Saccharomyces cerevisiae - genetics
Saccharomyces cerevisiae - metabolism
Spermatozoa - cytology
Spermatozoa - drug effects
Xenopus
ISSN:1940-6029, 1940-6029
Online Access:Get more information
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Summary:The detailed understanding of the DNA replication process requires structural insight. The combination of psoralen cross-linking 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 various forms of replication stress. The replication structures are stabilized in vivo (by psoralen cross-linking) prior to extraction and enrichment procedures, allowing their visualization at the transmission electron microscope. This chapter outlines the procedures required to visualize and interpret in vivo replication intermediates of genomic DNA, extracted from budding yeast, Xenopus egg extracts, or cultured mammalian cells.
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ISSN:1940-6029
1940-6029
DOI:10.1007/978-1-62703-706-8_15