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


Zellweger, Ralph; Lopes, Massimo (2018). Dynamic Architecture of Eukaryotic DNA Replication Forks In Vivo, Visualized by Electron Microscopy. In: Muzi-Falconi, Marco; Brown, Grant W. Genome Instability : Methods and Protocols. New York: Springer, 261-294.

Abstract

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.

Abstract

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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Additional indexing

Item Type:Book Section, refereed, original work
Communities & Collections:04 Faculty of Medicine > Institute of Molecular Cancer Research
07 Faculty of Science > Institute of Molecular Cancer Research
Dewey Decimal Classification:570 Life sciences; biology
610 Medicine & health
Scopus Subject Areas:Life Sciences > Molecular Biology
Life Sciences > Genetics
Language:English
Date:17 October 2018
Deposited On:25 Oct 2017 14:13
Last Modified:20 May 2024 03:46
Publisher:Springer
ISSN:1064-3745
ISBN:978-1-4939-7305-7
OA Status:Green
Publisher DOI:https://doi.org/10.1007/978-1-4939-7306-4_19
PubMed ID:29043630