DOUBLE-STRAND BREAKS AND UNTARGETED DNA METABOLIC EVENTS

双链断裂和非靶向 DNA 代谢事件

• 批准号: 6106566
• 负责人: MICHAEL 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/6106566
• 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 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.

工作总结：我们开发了一个系统， 研究位点特异性双链断裂的后果 (DSB)在S.啤酒。一 半乳糖诱导型HO-内切核酸酶切割位于半乳糖诱导型HO-内切核酸酶的 质粒（YZ-CEN）或YAC内的不同位置 含有人类DNA（YAC 12）。持久的、长寿命的DSB 会导致G-2级逮捕和死亡大多数特定地点的断裂 YAC被迅速修复，没有导致停止或死亡， 也没有在YZ-CEN质粒断裂时，它迅速 退化或修复。通过检查不同的菌株背景 我们已经提出了遗传控制的差异， 间接致死的证据一个持久的DSB在 含有YAC（VS 8）或YAC 12衍生物的λ DNA，u8 或u17在菌株LS 20中不诱导细胞周期停滞或致死。 然而，细胞周期阻滞和致死性都是由细胞周期阻滞引起的。 在其他菌株中持续存在DSB。因此，我们研究了是否 高拷贝酵母基因组文库或半乳糖- 诱导型人睾丸cDNA文库可导致间接致死 在LS 20中的持久DSB。七个酵母基因组片段， 已经鉴定了两种人cDNA，其增强了 持续的DSB一个克隆导致了SIR 4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 双链断裂末端连接。因此，SIR 4的新角色现在已经 被识别。当SIR 4不存在时，不可修复的DSB 在质粒或人工染色体中诱导， 与含有SIR 4的细胞相比，G2/M停滞延长。 虽然大多数SIR 4细胞最终分裂，但只有一个细胞分裂。 有限数量的细胞分裂和存活减少。的 SIR 4对未修复DSB耐受性的要求表明 染色质结构在检查点中起着重要作用， 适应持续的DNA损伤。