EFFECTS OF DNA LESIONS ON UNTARGETED DNA METABOLIC EVENTS

在DNA损伤后，酿酒酵母的细胞经历了 细胞周期G2期的RAD9依赖性停滞。 我们 试图阐明允许DNA损伤的分子机制， 发出对DNA损伤的整体细胞反应信号。 的效果 在45 bp YZ序列处酶促诱导的双链断裂（来自 MATYZ）在酵母单拷贝质粒的非酵母区中的表达。 考察 诱导持续的未修复的反式断裂导致 几乎完全丧失平板接种效率， - 是的 这种镀覆效率的损失部分地取决于镀覆层的厚度。 RAD9基因产物。 快速循环的单细胞检查 RAD+和rad9细胞群的非同步化， DSB表明，超过三分之一的细胞（包括未出芽的细胞和未出芽的细胞） [G-1]和出芽的[S = G-2]细胞）被抑制进一步的细胞 师. RAD+菌株的剩余细胞主要产生 含有永久性停滞的异常形状细胞的小菌落。 在rad9背景中未观察到小菌落，这解释了 该菌株的平板接种效率增加。 该系统提供了 用于研究DNA损伤的信号传导效应的手段，以及 设计调节细胞增殖的策略。 在一项使用 根据上述方法，我们研究了一个特定的工地DSB是否可以 用于物理映射酵母基因组内的特定基因。 我们有 构建并整合LEU2YZ、URA3YZ和RNC1YZLEU2靶向载体 进入酵母基因组。 整合后，将酵母转化为 用含有在gal下的HO内切酶基因的可选择质粒 对照半乳糖诱导的大小特异性染色体断裂在 染色体III和V上的LEU2YZ和URA3YZ整合位点 对应于这些基因的已知物理位置。 这些结果 表明该方法可以成功地用于物理映射 在酵母基因组内或在含有以下的YAC上克隆人或酵母基因： 人类DNA

Following DNA damage, cells of the yeast Saccharomyces cerevisiae undergo a RAD9-dependent arrest in the G2 phase of the cell cycle. We are attempting to elucidate the molecular mechanism that allows DNA lesions to signal global cellular responses to DNA damage. The effects of an enzymatically induced double-strand break at a 45 bp YZ sequence (from MATYZ) in a nonyeast region of a dispensable single copy plasmid were examined. Induction of a persistent unrepaired break in trans results in nearly complete loss of plating efficiency even though the plasmid is dispensable. This loss of plating efficiency is partially dependent on the RAD9 gene product. Examination of single cells from rapidly cycling unsynchronized populations of RAD+ and rad9 cells following induction of the DSB indicated that over one-third of the cells (including both unbudded [G-1] and budded [S = G-2] cells) were inhibited from further cell division. The remaining cells of a RAD+ strain give rise primarily to microcolonies containing permanently arrested aberrantly shaped cells. Microcolonies were not observed in the rad9 background which accounts for the increased plating efficiency of this strain. This system provides a means for studying the signalling effects of a DNA lesion as well as designing strategies for modulating cell proliferation. In a study using the above methodology, we have investigated whether a site specific DSB can be used to physically map specific genes within the yeast genome. We have constructed and integrated LEU2YZ, URA3YZ and RNC1YZLEU2 targeting vectors into the yeast genome. Following integration, the yeast were transformed with a selectable plasmid containing the HO endonuclease gene under gal control Galactose induced a size specific chromosomal fragmentation at the LEU2YZ and URA3YZ integration sites on chromosomes III and V respectively corresponding to the known physical location of these genes. These results indicate that this approach can be used successfully to physically map cloned human or yeast genes within the yeast genome or upon YACs containing human DNA.