EFFECTS OF DNA LESIONS ON UNTARGETED DNA METABOLIC EVENTS

一个未修复的和持续的DSB诱导在一个独特的YZ序列，在一个 单拷贝质粒（YZ-CEN）可导致小鼠的致死性， 酿酒酵母 调查间接后果 并了解它如何可能导致人类基因的改变 染色体材料，我们开发了一系列酵母人工 染色体（YACs）包含一个共同的360 kb的人类DNA片段 （YAC 12）和整合在各种人Alu序列处的URA 3-YZ序列 在YAC。这些菌株含有半乳糖诱导的HO-内切酶 在YZ站点产生了特定于站点的DSB。 对于大多数YAC（20/23） DSB的结果仅仅是URA 3标记的缺失， 整合的质粒DNA区域，可能通过重组 YZ断裂周围Alu序列之间的修复。然而，在三 在YAC中，DSB修复效率低下，导致持续性 DSB。 对于其中两个，间接致死性随后和YAC丢失 在大多数幸存者。对于第三个YAC，未观察到致死性; 然而，YAC通常会丢失。使用回撤程序， 将细胞在半乳糖中孵育并在不同时间加入葡萄糖， 我们研究了细胞死亡是否是持续性DSB的早期反应。 DSB诱导和断裂的致命后果之间的时间是 超过六个小时 对于持续性DSB未修复的YAC 并导致死亡，细胞进展有相当大的延迟， G-2快速修复的DSB不会导致细胞周期延迟， 这表明细胞周期检查点不会被激活， 是长寿的。我们建议，一个未修复的DSB必须以某种方式 由细胞解释。在几个YAC被修复后， 持久性DSB，大多数包含大的内部或末端 人类DNA中的缺失据观察，至少有一个 YAC，未修复的DSB不会导致死亡或细胞周期延迟， 这表明，解释一个中断可能部分取决于上下文。 我们试图了解细胞周期阻滞在G-2期的影响 间接致死性的影响。 我们已经确定，持续性YAC DSB的间接致死率是 非致命剂量的紫外线增强，导致G-2阻滞， 细胞 这种DSB单独不被细胞“感测”，因为它不 产生G-2逮捕或致命性。 调查G-2逮捕是否 为了满足YAC DSB的间接杀伤力要求，我们创建了一个 rad 9/rad 9缺失菌株，现在可以将多种致死性YAC 转化. 由于该rad 9/rad 9缺失突变体不经历G-2 DNA损伤后的逮捕，持续性DSB的影响（ 通常导致同基因RAD+菌株中的G-2停滞）现在可以 测定 我们还鉴定了一种酵母菌株（LS 20）， 在不可修复的质粒（YZ-CEN）中发生DSB后死亡，或 YAC（u8和u17）。 为了检测可能介导细胞凋亡的酵母基因， 携带YZ-CEN的LS 20， 用高拷贝酵母文库转化并通过复制筛选 与半乳糖和葡萄糖平板相关。 一个克隆（54-45）携带8个 已经鉴定了100 kb的酵母DNA，其显著增强了 HO诱导DSB后的致死率。

An unrepaired and persistent DSB induced at a unique YZ sequence in a dispensable single-copy plasmid (YZ-CEN) can result in lethality in the yeast Saccharomyces cerevisiae. To investigate the indirect consequences of a DSB and to understand how it might lead to alterations in human chromosomal material, we have developed a series of yeast artificial chromosomes (YACs) containing a common 360 kb fragment of human DNA (YAC12) and a URA3-YZ sequence integrated at various human Alu sequences in the YAC. The strains contained a galactose-inducible HO-endonuclease that produced a site specific DSB at the YZ site. For most YACs (20/23) the consequences of a DSB was simply the deletion of the URA3 marker and the integrated plasmid DNA region, presumably through recombinational repair between Alu sequences surrounding the YZ break. However, in three of the YACs the DSB was inefficiently repaired leading to a persistent DSB. For two of these, indirect lethality ensued and the YAC was lost in most of the survivors. For the third YAC, lethality was not observed; however, the YAC was usually lost. Using a pullback procedure in which cells are incubated in galactose and at various times glucose is added, we investigated if cell death was an early response to a persistent DSB. The time between DSB induction and the lethal consequences of a break was over six hours. For YACs in which the persistent DSB was not repaired and death resulted, there was considerable delay in cell progression at G-2. A DSB that was rapidly repaired did not lead to cell cycle delay, suggesting that a cell cycle checkpoint is not activated unless a break is long lived. We propose that an unrepaired DSB must somehow be interpreted by the cell. In the few YACs that were repaired following a persistent DSB, most contained either large internal or terminal deletions within the human DNA. The observation that, for at least one YAC, an unrepaired DSB does not lead to death or cell cycle delay, suggests that interpretation of a break may depend in part on context. We are attempting to understand the influence cell cycle arrest at G-2 has on the indirect lethality resulting from a DSB in dispensable DNA. We have determined that indirect lethality from a persistent YAC DSB is enhanced by nonlethal doses of UV that result in G-2 arrest of the cells. This DSB alone is not "sensed" by the cells since it does not produce G-2 arrest or lethality. To examine whether G-2 arrest is required for indirect lethality from a YAC DSB, we have created a rad9/rad9 deletion strain into which a variety of lethal YACs can now be transformed. Since this rad9/rad9 deletion mutant does not undergo G-2 arrest following DNA damage, the effects of a persistant DSB (that normally results in G-2 arrest in an isogenic RAD+ strain) can now be determined. We have also identified a yeast strain (LS20) that does not die following a DSB in a nonrepairable dispensable plasmid (YZ-CEN) or YACs (u8 and u17). In order to detect yeast genes that may mediate cell death following a DSB in dispensable DNA, LS20 carrying YZ-CEN was transformed with a high copy yeast library and screened by replica pronging to galactose and glucose plates. One clone (54-45) carrying 8 kb of yeast DNA has been identified which significantly enhances lethality following the HO-induced DSB.