Dynamic Architecture of Eukaryotic DNA Replication Forks In Vivo, Visualized by Electron Microscopy

The DNA replication process can be heavily perturbed by several different conditions of genotoxic stress, particularly relevant for cancer onset and therapy. The combination of psoralen crosslinking and electron microscopy has proven instrumental to reveal the fine architecture of in vivo DNA replic...

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Bibliographic Details
Published in:Methods in molecular biology (Clifton, N.J.) Vol. 1672; p. 261
Main Authors: Zellweger, Ralph, Lopes, Massimo
Format: Journal Article
Language:English
Published: United States 01.01.2018
Subjects:
Animals
Cross-Linking Reagents
DNA Replication
DNA, Fungal - ultrastructure
DNA, Single-Stranded - ultrastructure
Eukaryotic Cells - physiology
Microscopy, Electron
Nucleosomes - metabolism
Saccharomyces cerevisiae - genetics
Saccharomyces cerevisiae - metabolism
DNA replication
Nucleosome position
Replication fork reversal
In vivo replication intermediates
Electron microscopy
ssDNA
Psoralen crosslinking
ISSN:1940-6029, 1940-6029
Online Access:Get more information
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Summary:The DNA replication process can be heavily perturbed by several different conditions of genotoxic stress, particularly relevant for cancer onset and therapy. The combination of psoralen crosslinking and electron microscopy has proven instrumental to reveal the fine architecture of in vivo DNA replication intermediates and to uncover their remodeling upon specific conditions of genotoxic stress. The replication structures are stabilized in vivo (by psoralen crosslinking) 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 eukaryotic genomic DNA, and includes an improved method for enrichment of replication intermediates, compared to previously used BND-cellulose columns.
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ISSN:1940-6029
1940-6029
DOI:10.1007/978-1-4939-7306-4_19