DOUBLE-STRAND BREAKS AND UNTARGETED DNA METABOLIC EVENTS

双链断裂和非靶向 DNA 代谢事件

• 批准号: 6162096
• 负责人: M A RESNICK
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6162096
• 关键词: DNA damage; DNA repair; DNA replication; Escherichia coli; Saccharomyces cerevisiae; cell cycle; cell death; chromatin; complementary DNA; endonuclease; enzyme activity; fungal genetics; genetic markers; human genetic material tag; nucleic acid metabolism; nucleic acid sequence; plasmids; tissue /cell culture

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.

工作总结：我们开发了一个系统来检查结果 在YZ连接处的位点特异性双链断裂（DSB）， S.啤酒。 半乳糖诱导的HO- 核酸内切酶切割位于质粒（YZ-CEN）中或位于不同位置的YZ位点， 在含有人DNA的YAC（YAC 12）内的位置。一个持久的，长期的- 活的DSB导致G-2停滞和致命性。 这种间接杀伤力是 在遗传控制下，因为RAD 9的缺失导致更高的 生存能力。 YAC中的大多数位点特异性断裂迅速修复 并没有导致逮捕或死亡，也没有在YZ-CEN的突破， 当它被迅速降解或修复时，质粒。通过检查不同的 菌株背景，我们已经提出了遗传差异， 导致持久性DSB间接致死的对照。 一 含有YAC（VS 8）或YAC 12的λ DNA中的持续DSB 衍生物，u8或u17，不诱导细胞周期停滞或致死性， 菌株LS 20。然而，细胞周期阻滞和致死性都是由以下因素引起的： 在菌株NR 85（VS 8）和CBY（u8和u17）中存在持续的DSB。我们， 因此，研究是否存在高拷贝酵母基因组 文库或半乳糖诱导的人睾丸cDNA文库可以导致 LS 20中持续DSB的间接致死率。 七酵母基因组 已鉴定出增强致死性的片段和两种人类cDNA 持续的DSB。 一个酵母文库克隆已被拯救到 大肠杆菌中，测序并再转化到含有u8或 u17 YAC。 该克隆含有来自 包括沉默交配盒HMR的染色体III。我们提出 其作用原因是染色质可能在 信号。 我们现在已经确定，SIR 4基因， 在染色体沉默和染色质结构中发挥作用， 在对未修复的DSB的细胞反应中发挥作用。 这是 首次证明了染色质在细胞信号传导中的作用 DNA损伤。