Schmidt Kristina H, Pennaneach Vincent, Putnam Christopher D, Kolodner Richard D
Ludwig Institute for Cancer Research, University of North Texas, Health Science Center, Fort Worth, USA.
Methods Enzymol. 2006;409:462-76. doi: 10.1016/S0076-6879(05)09027-0.
Cells utilize numerous DNA metabolic pathways and cell-cycle checkpoints to maintain the integrity of their genome. Failure of these mechanisms can lead to genome instability, abnormal cell proliferation, and cell death. This chapter describes a method for the measurement of the rate of accumulating gross-chromosomal rearrangements (GCRs) in haploid cells of the yeast Saccharomyces cerevisiae. The isolation of cells with GCRs relies on the simultaneous loss of two counterselectable markers, CAN1 and URA3, within a nonessential region on the left arm of chromosome V. Healing of DNA breaks by de novo telomere addition, translocations, large interstitial deletions, and chromosome fusion has been detected using a PCR-based procedure for the mapping and amplification of breakpoint junctions, which is also described in detail here. This GCR analysis provides an effective tool for the assessment of the contribution by multiple cellular mechanisms to the maintenance of genome integrity.
细胞利用多种DNA代谢途径和细胞周期检查点来维持其基因组的完整性。这些机制的失效会导致基因组不稳定、异常细胞增殖和细胞死亡。本章描述了一种测量酿酒酵母单倍体细胞中总染色体重排（GCR）积累速率的方法。具有GCR的细胞的分离依赖于第五条染色体左臂非必需区域内两个反向选择标记CAN1和URA3的同时缺失。通过基于PCR的断点连接映射和扩增程序检测到了通过从头添加端粒、易位、大的中间缺失和染色体融合来修复DNA断裂的情况，本章也对此进行了详细描述。这种GCR分析为评估多种细胞机制对维持基因组完整性的贡献提供了一种有效工具。
