EFFECTS OF DNA LESIONS ON UNTARGETED DNA METABOLIC EVENTS

在一个独特的YZ序列诱导的未修复的和持久的DSB

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.