DOUBLE-STRAND BREAKS AND UNTARGETED DNA METABOLIC EVENTS

工作总结：我们已经开发了一个系统来检查后果 中YZ连接处的位点特异性双链断裂(DSB) 酿酒酵母体内可有可无的DNA。半乳糖诱导的HO- 核酸内切酶切割YZ位点，放置在一个质粒(YZ-CEN)或不同的 在含有人DNA的YAC中的位置(YAC12)。一个坚持不懈的，长久的- 活生生的DSB导致G-2被捕和死亡。这种间接的杀伤力是 在基因控制下，因为RAD9缺失导致更高 生存能力。YAC中的大多数特定部位的断裂都被迅速修复 没有导致逮捕或致死，YZ-CEN的破裂也没有 当它被快速降解或修复时。通过检查不同的 菌株背景我们已经提出了遗传上的差异 控制(S)对持续的DSB造成的间接致死负责。一个 含YAC(VS8)或YAC12的Lambda DNA中的持久性DSB U8或U17的衍生物不能诱导细胞周期停滞或致死。 菌株LS20。然而，细胞周期停滞和致命性都是由 在菌株NR85(VS8)和CBY(U8和U17)中发现一种持久性DSB。我们, 因此，检查了是否存在高拷贝的酵母基因组 文库或半乳糖诱导的人睾丸cDNA库可能导致 LS20中持续DSB的间接致命性。七种酵母基因组 已经确定了增强致命性的片段和两个人类cDNA 来自持久的DSB。一个酵母文库克隆已被营救到 大肠杆菌，测序并重新转化为LS20，其中包含U8或 U17 YAC。该克隆含有大约6kb的DNA，来自 第三号染色体包括沉默的交配盒HMR。我们建议说 其效果的原因是染色质可能在 信号。我们现在已经确定了SIR4基因，它可能 在染色体沉默和染色质结构中起作用，事实上 在细胞对未修复的DSB的反应中发挥作用。这是 染色质在细胞反应信号中的作用的首次证明 造成DNA损伤。

Summary of Work: We have developed a system to examine the consequences of a site-specific double-strand break (DSB) at a YZ junction in dispensable DNA within S. cerevisiae. A galactose-inducible HO- endonuclease cuts a YZ site placed in a plasmid (YZ-CEN) or at various positions within a YAC containing human DNA (YAC12). A persistent, long- lived DSB led to G-2 arrest and lethality. This indirect lethality is under genetic control since deletion of RAD9 resulted in higher viability. Most site-specific breaks in the YACs were rapidly repaired and did not lead to arrest or lethality, nor did a break in the YZ-CEN plasmid when it was rapidly degraded or repaired. By examining different strain backgrounds we have suggested differences in the genetic control(s) responsible for indirect lethality from a persistent DSB. A persistent DSB in a lambda DNA containing YAC (VS8) or the YAC12 derivatives, u8 or u17, did not induce cell cycle arrest or lethality in strain LS20. However, both cell cycle arrest and lethality resulted from a persistent DSB in strains NR85 (VS8) and CBY (u8 and u17). We, therefore, examined whether the presence of a high-copy yeast genomic library or a galactose-inducible human testis cDNA library could lead to indirect lethality by a persistent DSB in LS20. Seven yeast genomic fragments and two human cDNAs have been identified that enhance lethality from a persistent DSB. One yeast library clone has been rescued into E.coli, sequenced and retransformed into LS20 containing either the u8 or u17 YACs. This clone contained approximately 6 kb of DNA from chromosome III including the silent mating cassette HMR. We proposed that the reason for its effect is that chromatin may play an important role in the signaling. We have now established that the SIR4 gene, which may play a role in chromosome silencing and chromatin structure, does in fact play a role in the cellular response to the unrepaired DSB. This is the first demonstration of a role for chromatin in cell signaling in response to DNA damage.