DOUBLE STRAND BREAK MUTAGENESIS: TRANSCRIPTION AND P53

双链断裂诱变：转录和 P53

• 批准号: 6342223
• 负责人: HOWARD L LIBER
• 金额: $ 21.79万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-01-26 至 2002-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6342223
• 关键词: DNA damage; Saccharomyces cerevisiae; X ray; apoptosis; cell cycle; cell line; flow cytometry; gene mutation; gene targeting; genetic transcription; lymphoblast; p53 gene /protein; radiation genetics; restriction endonucleases; site directed mutagenesis; thymidine kinase; transfection; transfection /expression vector

This work will investigate transcription, and the p53-directed response to DNA damage, as factors that affect the processing of double strand breaks (DSB) into mutations. The work is based is based being able to specifically target restriction sites for the extremely rare-cutting yeast SceI nuclease to the endogenous thymidine kinase (tk) gene in human lymphoblast cells. The mutagenic and apoptotic effects of DSB induced at those sites can be determined. Hypothesis to be explored are as follows. (i) DSB are more effective at inducing mutation or apoptosis when occurring in a gene undergoing transcription than in a non-transcribed region. (ii) DSB are much more efficient mediators of deletion mutations in p53 mutant cells. (iii) Two DSB within a few kilobases will have synergistic effects. (iv) X-rays will act synergistically with DSB to lead to large-scale mutations. (v) Recombination between Alu sequences will not stimulate mutagenesis in p53 wild type cells, but they may in p53 mutants. (vi) Apoptosis will not decrease mutagenesis unless a major premutagenic lesion is also a key signal for apoptosis. Aim 1 will determine the effect of specific DSB on mutagenesis in the tk gene and on apoptosis. Lymphoblast lines will be constructed that will respond to an exogenous inducer by transcribing the SceI nuclease. Second, gene targeting vectors will be constructed and utilized to introduce SceI restriction sites to distinct positions in the active tk allele of TK6 heterozygotes. The effectiveness of nuclease-generated DSB alone or with a dose of ionizing radiation in inducing gene mutation and apoptosis will be determined. Aim 2 will examine th4e effect of transcription level on mutagenesis and apoptosis induced by DSB. Studies will indicate whether cells actively transcribing a gene with a DSB are more sensitive to mutations in that gene and/or to apoptosis. A tk transactivator will be added to the cells, allowing levels of transcription of tk RNA to be modulated. Other experiments will explore the relationship between cycle position and mutagenesis. Aim 3 will examine the effect of p53 status on apoptosis and mutagenesis induced by DSB. Cells will be transfected with a dominant-0negative p53, and the effects on mutagenesis and apoptosis, under normal and high levels of transcription.

这项工作将研究转录以及 p53 对 DNA 损伤的定向反应，作为影响双链断裂 (DSB) 加工成突变的因素。这项工作的基础是能够将极其罕见的切割酵母 SceI 核酸酶的限制性位点特异性靶向人淋巴母细胞中的内源胸苷激酶 (tk) 基因。可以确定 DSB 在这些位点诱导的诱变和细胞凋亡效应。待探讨的假设如下。 (i) 当发生在正在进行转录的基因中时，DSB 比发生在非转录区域中更能有效地诱导突变或凋亡。 (ii) DSB 是 p53 突变细胞中更有效的缺失突变介体。 (iii) 几千碱基以内的两个 DSB 将产生协同效应。 (iv) X射线将与DSB协同作用，导致大规模突变。 (v) Alu 序​​列之间的重组不会刺激 p53 野生型细胞中的诱变，但可能会刺激 p53 突变体细胞中的诱变。 (vi) 细胞凋亡不会减少诱变，除非主要的诱变前病变也是细胞凋亡的关键信号。目标 1 将确定特定 DSB 对 tk 基因诱变和细胞凋亡的影响。将构建通过转录 SceI 核酸酶对外源诱导物作出反应的淋巴细胞系。其次，将构建基因靶向载体并利用其将 SceI 限制性位点引入到 TK6 杂合子的活性 tk 等位基因的不同位置。将确定核酸酶产生的 DSB 单独或与一定剂量的电离辐射一起诱导基因突变和细胞凋亡的有效性。目标 2 将检查转录水平对 DSB 诱导的诱变和细胞凋亡的影响。研究将表明，主动转录带有 DSB 的基因的细胞是否对该基因的突变和/或细胞凋亡更敏感。将向细胞中添加 tk 反式激活因子，从而调节 tk RNA 的转录水平。 其他实验将探讨循环位置和诱变之间的关系。目标 3 将检查 p53 状态对 DSB 诱导的细胞凋亡和诱变的影响。细胞将被显性 0 阴性 p53 转染，并在正常和高水平转录下对诱变和凋亡产生影响。