DOUBLE STRAND BREAK MUTAGENESIS: TRANSCRIPTION AND P53

这项工作将调查转录，和p53-导向的DNA损伤的反应，作为影响双链断裂（DSB）到突变的加工的因素。这项工作是基于能够特异性靶向限制性位点的极其罕见的切割酵母SceI核酸酶的内源性胸苷激酶（tk）基因在人类淋巴母细胞。可以确定在这些位点诱导的DSB的致突变和凋亡作用。待探讨的假设如下。(i)当DSB发生在经历转录的基因中时比发生在非转录区域中时更有效地诱导突变或凋亡。(ii)DSB是p53突变细胞中缺失突变的更有效的介体。(iii)两种DSB在几种酶的作用下会产生协同效应。(iv)X射线将与DSB协同作用，导致大规模突变。(v)Alu序列之间的突变在p53野生型细胞中不会刺激诱变，但在p53突变体中可能。(vi)细胞凋亡不会减少诱变，除非主要的诱变前损伤也是细胞凋亡的关键信号。目的1将确定特定DSB对tk基因突变和细胞凋亡的影响。将构建淋巴母细胞系，其将通过转录SceI核酸酶对外源诱导物作出响应。其次，将构建基因靶向载体并用于将SceI限制性位点引入TK 6杂合子的活性tk等位基因中的不同位置。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。目的2探讨转录水平对DSB致突变和诱导细胞凋亡的影响。研究将表明是否细胞积极转录基因与DSB更敏感的基因突变和/或细胞凋亡。将tk反式激活因子添加到细胞中，允许调节tk RNA的转录水平。 其他实验将探索环位置和诱变之间的关系。目的3研究p53状态对DSB诱导的细胞凋亡和突变的影响。将用显性负性p53转染细胞，并在正常和高水平转录下观察对诱变和凋亡的影响。

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.