Lengronne A, Pasero P, Bensimon A, Schwob E
Institute of Molecular Genetics, CNRS UMR 5535 and Université Montpellier II, 1919 Route de Mende, 34293 Montpellier Cedex 5, France.
Nucleic Acids Res. 2001 Apr 1;29(7):1433-42. doi: 10.1093/nar/29.7.1433.
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.
真核生物染色体复制从众多起始点开始，其激活受细胞周期和检查点机制的时间控制。酵母在确定DNA复制起始所需的遗传元件方面非常有用，但在这种模式生物中缺乏简单而精确的监测S期进程的工具。在这里，我们描述了一种TK(+)酵母菌株和允许将外源BrdU掺入基因组DNA的条件，以及在酵母细胞核或梳理的DNA分子上检测DNA合成位点的方案。通过定量总酵母DNA或单个染色体上的BrdU来监测S期进程。使用这些工具，我们表明酵母染色体同步复制，并且DNA合成发生在离散的亚核焦点处。对来自羟基脲阻滞细胞的单个DNA分子上的BrdU信号分析表明，复制叉在平均相距46 kb的起始点8-9 kb处停滞。总BrdU掺入量的定量表明，在这些细胞中有190个“早期”起始点已启动，并且晚期复制区域可能占酵母基因组的40%。更一般地说，这里概述的方法将有助于理解野生型酵母中DNA复制的动力学，并完善几种突变体的表型。
