Monitoring S phase progression globally and locally using BrdU incorporation in TK+ yeast strains

Eukaryotic chromosome replication is initiated from numerous origins and it s activation is temporally controlled by cell cycle and checkpoint mechanis ms. Yeast has been very useful in defining the genetic elements required fo r 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 in to genomic DNA, along with protocols to detect the sites of DNA synthesis i n 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 a nd 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 apar t on average. Quantification of total BrdU incorporation suggests that 190 'early' origins have fired in these cells and that late replicating territo ries might represent up to 40% of the yeast genome. More generally, the met hods outlined here will help understand the kinetics of DNA replication in wild-type yeast and refine the phenotypes of several mutants.