MOLECULAR ANALYSIS OF X-RAY DAMAGE AND REPAIR IN YEAST

酵母 X 射线损伤和修复的分子分析

• 批准号: 2175974
• 负责人: DAVID SCHILD
• 金额: $ 10.27万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-03-01 至 1995-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2175974
• 关键词: DNA repair; Saccharomyces; X ray; chromosome aberrations; fungal genetics; gene mutation; gene rearrangement; genetic mapping; genetic recombination; genetic strain; genetic transcription; haploidy; molecular cloning; molecular genetics; mutant; nucleic acid sequence; protein purification; pulsed field gel electrophoresis; radiation dosage; radiation genetics; radiation sensitivity; recombinant DNA; yeasts

In the yeast Saccharomyces cerevisiae, recombination plays a central role in the repair of DNA double-strand breaks (DSB). Research on X-ray induced DSB and repair in yeast will be continued, making use of the well characterized radiation- sensitive (rad) mutants that we have previously studied. Several different experimental approaches are proposed which all address basic aspects of recombinational repair. Recent pulsed-field gel electrophoresis (PFGE) techniques (Mortimer et al., 1991) will be used to investigate the molecular basis of recombinational repair of DSB. As described earlier, diploid yeast strains that contain one circular and one linear derivative of chromosome III can be used in combination with PFGE to measure the frequency of DSB, and to separately assay molecules that have undergone sister-chromatid exchange (SCE) and those that are products of recombination between homologous chromosomes. Thus, the relative importance in repair of SCE versus interhomolog recombination can be assessed. Several other significant questions about repair will be addressed, for example whether DSB can be repaired by non-recombinational mechanisms in yeast and whether one unrepaired DSB corresponds to a lethal event. The molecular phenotypes of wild-type and rad mutant strains will be characterized using PFGE and molecular endpoints will be related to biological events, including survival, recombination and chromosome loss. We also hope to develop further assays for SCE and for single-strand nicks in yeast using circular chromosomes. In related studies, PFGE will be used to characterize chromosomal aberrations induced by X-rays in yeast. There are extensive studies of such aberrations in higher eukaryotes, but few in lower eukaryotes because of the difficulty of observing aberrations cytologically. PFGE now enables us to detect these aberrations, including translocations and large deletions, and available rad mutants will be used to ask whether recombinational repair plays a role in their formation. The frequency of various types of aberration versus X-ray dose will be determined, both for wild-type strains and for strains mutant in each of the two major types of X-ray repair in yeast. If recombinational repair is important in the formation of aberrations, few such aberrations are expected in mutants blocked in this process. The molecular analysis of genes required for recombinational repair will be continued. The DNA sequencing of genes we have cloned will be completed by finishing the sequencing of the RAD51 and RAD55 genes. We will also focus on the regulation of the RAD51 and RAD54 genes and on obtaining the RAD54 protein. RAD51 and RAD54 possess a common upstream sequence and it will be determined if this is required for their observed induction by DNA damage. These genes will also be tested for cell-cycle regulation. Antibodies against part of the RAD54 protein will be affinity purified and used to assay induced and constitutive levels of this protein, to study its intracellular localization and to purify the intact protein.

在酿酒酵母的酵母菌中，重组发挥 DNA双链断裂（DSB）的修复中的中心作用。 X射线诱导的DSB和酵母修复的研究将是 继续，利用良好特征的辐射 - 我们以前已经研究过的敏感（RAD）突变体。 一些 提出了不同的实验方法 重组维修的基本方面。 最新的脉冲场凝胶电泳（PFGE）技术（Mortimer 等，1991）将用于研究 DSB的重组维修。 如前所述，二倍体酵母 包含一个圆形和一个线性衍生物的菌株 染色体III可以与PFGE结合使用以测量 DSB的频率，并分别测定具有的分子 接受了姐妹 - 染色剂交换（SCE）和那些 同源染色体之间的重组产物。 因此， SCE与间的修复方面的相对重要性 可以评估重组。 其他几个重要问题 关于维修将被解决，例如DSB是否可以 通过酵母中的非重组机制修复 未修复的DSB对应于致命事件。 分子 野生型和rad突变菌株的表型将是 使用PFGE和分子端点的特征将是相关的 进行生物事件，包括生存，重组和 染色体损失。 我们也希望为SCE开发进一步的测定法 并使用圆形染色体在酵母中的单链刻痕。 在相关研究中，PFGE将用于表征染色体 X射线在酵母中引起的畸变。 有很多 对较高真核生物中这种畸变的研究，但很少 真核生物由于难以观察畸变 在细胞学上。 PFGE现在使我们能够检测到这些畸变， 包括易位和大删除以及可用的RAD 突变体将用于询问是否进行重组维修 在他们的形成中的作用。 各种类型的频率 将确定畸变与X射线剂量，均用于野生型 两种主要类型的每一种 X射线维修酵母。 如果重组维修在 畸变的形成，预计很少发生这种畸变 突变体在此过程中被阻塞。 重组修复所需的基因的分子分析 将继续。 我们克隆的基因的DNA测序 通过完成RAD51和RAD55的测序完成完成 基因。 我们还将专注于Rad51和Rad54的调节 基因并获得Rad54蛋白。 Rad51和Rad54拥有 一个常见的上游序列，将确定是否是 通过DNA损伤观察到的诱导所必需。 这些基因 还将测试细胞周期调节。 反对的抗体 RAD54蛋白的一部分将是亲和力纯化的，用于 测定该蛋白的诱导和组成水平，以研究 它的细胞内定位并纯化完整的蛋白质。