DOUBLE STRAND BREAK MUTAGENESIS: TRANSCRIPTION AND P53

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）转化为突变的因素。这项工作的基础是能够特异性地靶向人类淋巴母细胞内源性胸苷激酶（tk）基因中极为罕见的酵母SceI核酸酶的限制性位点。可以确定DSB在这些位点诱导的致突变和凋亡作用。需要探讨的假设如下。(1)与非转录区相比，发生在正在转录的基因中的DSB在诱导突变或凋亡方面更有效。（ii） DSB是p53突变细胞中更有效的缺失突变介质。在几千碱基内的两个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序列之间的重组在p53野生型细胞中不会刺激突变，但在p53突变体中可能会。（vi）细胞凋亡不会减少诱变，除非一个主要的诱变前病变也是细胞凋亡的关键信号。目的1将确定特异性DSB对tk基因突变和细胞凋亡的影响。将构建淋巴母细胞系，通过转录SceI核酸酶对外源性诱导剂产生反应。其次，构建基因靶向载体，将SceI酶切位点引入TK6杂合子活性tk等位基因的不同位置。核酸酶产生的DSB单独或与一定剂量的电离辐射诱导基因突变和细胞凋亡的有效性将被确定。目的2研究转录水平对DSB诱变和凋亡的影响。研究将表明，活跃转录带有DSB的基因的细胞是否对该基因的突变和/或凋亡更敏感。tk转录激活剂将被添加到细胞中，允许tk RNA的转录水平被调节。其他实验将探索周期位置与诱变之间的关系。目的3研究p53状态对DSB诱导的细胞凋亡和诱变的影响。在正常和高水平的转录下，细胞将被转染显性0负p53，并对突变和凋亡产生影响。

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.