Nucleic Acids Research, 2001, Vol. 29, No. 7 1433-1442
© 2001 Oxford University Press
Monitoring S phase progression globally and locally using BrdU incorporation in TK+ yeast strains
Institute of Molecular Genetics, CNRS UMR 5535 and Université Montpellier II, 1919 Route de Mende, 34293 Montpellier Cedex 5, France and 1Institut Pasteur, Département des Biotechnologies, 25 Rue du Dr Roux, 75724 Paris Cedex 15, France
Eukaryotic chromosome replication is initiated from numerous origins and its activation is temporally controlled by cell cycle and checkpoint mechanisms. Yeast has been very useful in defining the genetic elements required for initiation of DNA replication, but simple and precise tools to monitor S phase progression are lacking in this model organism. Here we describe a TK+ yeast strain and conditions that allow incorporation of exogenous BrdU into genomic DNA, along with protocols to detect the sites of DNA synthesis in yeast nuclei or on combed DNA molecules. S phase progression is monitored by quantification of BrdU in total yeast DNA or on individual chromosomes. Using these tools we show that yeast chromosomes replicate synchronously and that DNA synthesis occurs at discrete subnuclear foci. Analysis of BrdU signals along single DNA molecules from hydroxyurea-arrested cells reveals that replication forks stall 8–9 kb from origins that are placed 46 kb apart on average. Quantification of total BrdU incorporation suggests that 190 ‘early’ origins have fired in these cells and that late replicating territories might represent up to 40% of the yeast genome. More generally, the methods outlined here will help understand the kinetics of DNA replication in wild-type yeast and refine the phenotypes of several mutants.
* To whom correspondence should be addressed. Tel: +33 467 61 36 79; Fax: +33 467 04 02 31; Email: schwob{at}igm.cnrs-mop.fr
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