EFFECTS OF DNA LESIONS ON UNTARGETED DNA METABOLIC EVENTS

在DNA损伤后，酿酒酵母细胞经历了 依赖RAD9的细胞周期的G2期停滞。我们是 试图阐明允许DNA损伤 发出全球细胞对DNA损伤的反应信号。网络安全的影响 酶诱导的45bp YZ序列上的双链断裂(来自 MATYZ)在可有可可单拷贝质粒非酵母区 检查过了。在TRANS中诱导永久性未修复的断裂会导致 几乎完全丧失了平板效率，即使质粒是 可有可无。这种电镀效率的损失部分取决于 RAD9基因产物。快速循环中单细胞的检测 RAD和RAD9细胞在诱导后的不同步群体 DSB表示，超过三分之一的细胞(包括两个未萌发的细胞 [G-1]和萌发的[S=G-2]细胞)被进一步抑制 组织。RAD菌株的剩余细胞主要产生 含有永久停止的形状异常的细胞的微集落。 在rad9背景中没有观察到微集落，这解释了 提高了该菌株的培养效率。该系统提供了一种 研究DNA损伤的信号效应的方法以及 设计调控细胞增殖的策略。在一项研究中使用 在上述方法中，我们调查了特定于站点的DSB是否可以 用于物理定位酵母基因组中的特定基因。我们有 构建并整合了LEU2YZ、URA3YZ和RNC1YZLEU2靶向载体 进入酵母基因组。在整合后，酵母被转化为 在GAL下与含有HO内切酶基因的可选择质粒连接 对照半乳糖引起大小特定的染色体断裂 染色体III和V上的LEU2YZ和URA3YZ整合位点 与这些基因的已知物理位置相对应。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包含以下内容的YAC上克隆人或酵母基因 人类的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.