MOLECULAR ANALYSIS OF X-RAY DAMAGE AND REPAIR IN YEAST

在酿酒酵母中，重组发挥了作用 在DNA双链断裂(DSB)修复中的核心作用。 X射线诱导酵母菌DSB及其修复的研究 继续，利用具有良好特性的辐射- 我们之前研究过的敏感(Rad)突变体。几个 提出了不同的实验方法，这些方法都解决了 重组修复的基本方面。 最新脉冲场凝胶电泳(PFGE)技术(Mormer 等人，1991)将被用来研究 DSB的重组修复。如前所述，二倍体酵母 包含一个圆形和一个线性导数的菌株 三号染色体可与PFGE结合使用来测量 DSB的频率，并分别检测具有 接受姐妹染色单体交换(SCE)的和那些 同源染色体间重组的产物。因此， 姐妹染色单体互换修复与同源异体修复的相对重要性 重组是可以评估的。其他几个重要问题 将解决有关修复的问题，例如DSB是否可以 在酵母中被非重组机制修复，以及是否有 未修复的DSB对应于致命事件。分子 野生型和rad突变株的表型将是 使用PFGE表征和分子终点将相关 生物事件，包括生存、重组和 染色体丢失。我们还希望进一步开发姐妹染色单体交换的检测方法。 对于使用环形染色体的酵母中的单链缺口。 在相关研究中，脉冲电场凝胶电泳法将用于鉴定染色体 X射线在酵母中诱发的畸变率。有广泛的 在高等真核生物中对这种畸变的研究，但在低等真核生物中很少 真核生物，因为很难观察到畸变 细胞学上。PFGE现在使我们能够检测到这些像差， 包括移位和大片段缺失，以及可用的辐射 突变体将被用来询问重组修复是否起作用 在它们的形成过程中扮演了一个角色。各种类型的频率 将确定像差与X射线剂量的关系，这两种类型均为野生型 菌株和在两种主要类型中每一种都发生突变的菌株 酵母菌中的X射线修复。如果重组修复在 像差的形成，很少有这样的像差在 突变体在这一过程中受阻。 重组修复所需基因的分子分析 将会继续。我们已经克隆的基因的DNA测序将 完成RAD51和RAD55的测序 基因。我们还将重点关注RAD51和RAD54的监管 基因和获得RAD54蛋白。RAD51和RAD54具有 一个常见的上游序列，并将确定这是否是 它们被观察到的DNA损伤诱导所需的。这些基因 还将接受细胞周期调节的测试。抗病毒抗体 部分RAD54蛋白将被亲和纯化并用于 检测该蛋白的诱导和组成水平，以研究 它在细胞内的定位和纯化完整的蛋白。

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.