Peng Jie, Raghuraman M K, Feng Wenyi
Department of Biochemistry & Molecular Biology, SUNY Upstate Medical University, 4287 Weiskotten Hall, 750 East Adams Street, Syracuse, NY, 13210, USA.
Methods Mol Biol. 2014;1170:501-15. doi: 10.1007/978-1-4939-0888-2_27.
DNA replication defects are an underlying cause of genome instability, which could stem from alterations in replication intermediates such as extensive single-stranded DNA (ssDNA). Under replication stress, ssDNA is a precursor of the ultimate double-strand breaks (DSBs). Indeed, mutations that render the cell incapable of mediating and protecting the replication forks produce ssDNA genome-wide at high frequency and cause lethality when encountering DNA damage or replication perturbation. Here we describe two related microarray-based methods to query genetically unstable yeast cultures, such as the mec1 and rad53 mutants. These mutants are defective in central protein kinases in the checkpoint pathway. To induce ssDNA and DSB formation in these mutants, we utilize hydroxyurea, a drug that causes nucleotide shortage in the cell.
DNA复制缺陷是基因组不稳定的一个潜在原因，其可能源于复制中间体的改变，如广泛的单链DNA（ssDNA）。在复制应激下，ssDNA是最终双链断裂（DSB）的前体。事实上，使细胞无法介导和保护复制叉的突变会在全基因组范围内高频产生ssDNA，并在遇到DNA损伤或复制干扰时导致细胞死亡。在这里，我们描述了两种基于微阵列的相关方法，用于检测基因不稳定的酵母培养物，如mec1和rad53突变体。这些突变体在检查点途径中的核心蛋白激酶方面存在缺陷。为了在这些突变体中诱导ssDNA和DSB的形成，我们使用羟基脲，一种导致细胞内核苷酸短缺的药物。
