DOUBLE-STRAND BREAKS AND UNTARGETED DNA METABOLIC EVENTS

工作总结：我们已经开发了一个系统来 检查特定部位双链断裂的后果 (DSB)位于酿酒酵母可支配DNA的YZ连接处。一个 半乳糖诱导的HO-内切酶切割放置在 质粒(YZ-CEN)或在YAC内的不同位置 含有人类DNA(YAC12)。一个持久的、长寿的DSB 可能导致G-2被捕和死亡。大多数特定于站点的中断在 YAC得到了迅速修复，没有导致逮捕或死亡， 当YZ-CEN质粒迅速断裂时，也没有 降级的或修复的。通过检查不同的菌株背景 我们提出了不同的基因控制(S)负责 来自持久的DSB的间接致命性。对象中的持久性DSB 含有YAC(VS8)或YAC12衍生物U8的Lambda DNA 或u17，对LS20菌株没有诱导细胞周期停滞或致死作用。 然而，细胞周期停滞和致命性都是由 其他菌株中的持久性DSB。因此，我们研究了 高拷贝酵母基因组文库或半乳糖的存在- 诱导型人睾丸cdna文库可导致间接致死 由LS20中的持久DSB执行。七个酵母基因组片段和 已发现两个人类cDNA，它们可以增强人的致命性 一个持久的DSB。一个克隆导致了SIR4的鉴定 信号反应中的基因。酵母中的这种基因可以影响 转录沉默、染色质组织、衰老和 双链断裂末端相连。因此SIR4现在有了一个新的角色 已被确认身份。当SIR4缺失时，无法修复的DSB 在可有可无的质粒或人造染色体中诱导导致 与含有SIR4的细胞相比，G2/M期停滞时间延长。 虽然大多数SIR4细胞最终会分裂，但只有一个 细胞分裂数量有限，存活率降低。这个 SIR4对未修复DSB容忍度的要求建议 染色质结构在检查点中起着重要作用 适应持续的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 can lead to G-2 arrest and lethality. 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 other strains. 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 clone led to the identification of the SIR4 gene in the signaling response. This gene in yeast can affect transcriptional silencing, chromatin organization, aging and double-strand break endjoining. Thus a new role for SIR4 has now been identified. When SIR4 is absent, the unrepairable DSB induced in a dispensable plasmid or artificial chromosome leads to prolonged G2/M arrest as compared to cells containing SIR4. While most of the SIR4 cells eventually divide, there is only a limited number of cell divisions and survival is reduced. The requirement of SIR4 for toleration of an unrepaired DSB suggests that chromatin structure plays an important role in checkpoint adaptation to a persistent DNA lesion.