Efficient duplication of eukaryotic genomes relies on the sequential activation of thousands of replication origins distributed along the chromosomes. This process follows a complex spatial and temporal program, which is under the control of epigenetic mechanisms and is tightly linked to the functional organization of the nucleus (Méchali, 2001). Recent evidence indicates that this replication program is also controlled by checkpoint kinases that monitor the correct execution of S phase, such as ATR in human and Mec1 in budding yeast (Tourriere and Pasero, 2007). Over the last twenty years, a wide variety of methods has been developed to map replication origins in eukaryotic cells (DePamphilis, 1997). These techniques have shown that eukaryotic genomes contain an excess of potential replication origins and that only a fraction of these origins fire within a given S phase. However, the dynamics of DNA replication remains poorly defined at the level of individual chromosomes, essentially because biochemical assays provide averaged replication profiles for a population of cells.
The recent development of single-molecule assays has shed new light on the dynamics of DNA replication at the level of individual chromosomes. These techniques have been successfully used to monitor DNA replication in a variety of organisms, including bacteria, yeasts, xenopus and mammals (Anglana et al., 2003; Breier et al., 2005; Herrick et al., 2000; Jackson and Pombo, 1998; Lemaitre et al., 2005; Pasero et al., 2002; Patel et al., 2006). Here, we describe the analysis of DNA replication in S. cerevisiae by DNA combing, one of the most widely used single-molecule assay (Bensimon et al., 1994; Michalet et al., 1997).
In this assay, replication origins are first labeled with bromodeoxyuridine (BrdU) in early S phase. Chromosomal DNA is then purified in agarose plugs and stretched on silanized coverslips. This procedure generates long, parallel DNA fibers, with a uniform extension of 2 kb/μm. Newly-replicated DNA is then detected with a monoclonal antibody directed against BrdU and DNA fibers are counterstained with an antibody against single-stranded DNA. Replicating DNA fibers are revealed with fluorescent secondary antibodies and are imaged with an epifluorescence microscope coupled to a CCD camera. Representative examples of large DNA fibers (>400 kb) are shown in Figure 1. From these images, a large number of replication parameters can be derived, such as the rates of initiation and elongation and the percentage of substitution for individual DNA fibers. When combined with fluorescence in situ hybridization, this technique allows a precise mapping of active origins along a chromosome of interest (Anglana et al., 2003; Lebofsky et al., 2006; Pasero et al., 2002). DNA combing can also be used to monitor fork recovery after a replication stress, using a combination of halogenated nucleotides (CldU/IdU) that can be distinguished with specific anti-BrdU antibodies (Luke et al., 2006; Tourriere et al., 2005).
The protocol described below is meant to detect BrdU incorporation in yeast cells arrested in early S phase with hydroxyurea (HU). It can be easily adapted to monitor ongoing DNA replication in asynchronous yeast cultures or to analyze DNA replication in mammalian cells. Specific details regarding the preparation of BrdU-labeled genomic DNA from mammalian cells are available upon request.
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