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J Mol Biol. 2013 Nov 29;425(23):4845-55. doi: 10.1016/j.jmb.2013.03.040. Epub 2013 Apr 1.

Replication dynamics: biases and robustness of DNA fiber analysis.

Author information

1
Institut Curie, Centre de Recherche, 26 rue d'Ulm, 75248 Paris Cedex 05, France; Université Pierre et Marie Curie Paris 06, 4 Place Jussieu, 75005 Paris, France; Centre National de la Recherche Scientifique, Unité Mixte de Recherche 3244, 75248 Paris Cedex 05, France.

Abstract

The factors that govern replication programs are still poorly identified in metazoans, especially in mammalian cells. Thanks to molecular combing, the dynamics of DNA replication can be assessed at the genome-scale level from the cumulative analysis of single DNA fibers. This technique notably enables measurement of replication fork speed and fork asymmetry and that of distances separating either initiation or termination events. The results presented here aim to evaluate requirements critical to accurate measurement of replication parameters by molecular combing. We show that sample size, fiber length and DNA counterstaining are crucial to gain robust information concerning replication dynamics. Our results thus provide a methodological frame to investigate the DNA replication program through molecular combing analyses.

KEYWORDS:

CHEF; Chinese hamster embryonic fibroblast; DNA combing; IOD; ITD; MEF; fork asymmetry; inter-origin distance; inter-termination distance; mouse embryonic fibroblast; replication fork speed

PMID:
23557832
DOI:
10.1016/j.jmb.2013.03.040
[Indexed for MEDLINE]

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