title: Single-Molecule Analysis of Replicating Yeast Chromosomes.
abstract: The faithful replication of eukaryotic chromosomal DNA occurs during S phase once per cell cycle. Replication is highly regulated and is initiated at special structures, termed origins, from which replication forks move out bidirectionally. A wide variety of techniques have been developed to study the features and kinetics of replication. Many of these, such as those based on flow cytometry and two-dimensional and pulsed-field gel electrophoresis, give a population-level view of replication. However, an alternative approach, DNA fiber analysis, which was originally developed more than 50 years ago, has the advantage of revealing features of replication at the level of individual DNA fibers. Initially based on autoradiography, this technique has been superseded by immunofluorescence-based detection of incorporated halogenated thymidine analogs. Furthermore, derivations of this technique have been developed to distribute and stretch the labeled DNA fibers uniformly on optically clear surfaces. As described here, one such technique-DNA combing, in which DNA is combed onto silanized coverslips-has been used successfully to monitor replication fork progression and origin usage in budding yeast.
authors.affiliation: Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada; Donnelly Centre, University of Toronto, Toronto, Ontario M5S 3E1, Canada.
authors.forename: David
authors.initials: D
authors.lastname: Gallo
authors.affiliation: Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada; Donnelly Centre, University of Toronto, Toronto, Ontario M5S 3E1, Canada.
authors.forename: Gang
authors.initials: G
authors.lastname: Wang
authors.affiliation: Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada; Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario M5S 3E5, Canada; Donnelly Centre, University of Toronto, Toronto, Ontario M5S 3E1, Canada.
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Yip
authors.affiliation: Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada; Donnelly Centre, University of Toronto, Toronto, Ontario M5S 3E1, Canada.
authors.forename: Grant W
authors.initials: GW
authors.lastname: Brown
mesh_terms: D002455:Cell Division; D015825:Chromosomes, Fungal; D004261:DNA Replication; D008856:Microscopy, Fluorescence / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000502:physiology*