DNA REPLICATION, REPAIR, AND MUTAGENESIS IN EUKARYOTIC AND PROKARYOTIC CELLS

突变研究主要集中在大肠杆菌UmuDC蛋白上。大肠杆菌或同源物 (e.g., MucAB）。 荧光分析已被用于 分析UmuD到致突变活性形式（UmuD '）的加工。 在 e.大肠杆菌，这一过程是低效的，需要去阻遏水平的 激活RecA用于UmuD切割。 我们已经检测到一种UmuD样蛋白， 多种肠杆菌几个非突变株UmuD缺陷 处理，而可变性差的S.鼠伤寒，令人惊讶的是， 最有效的。 为了研究MucA裂解，我们过量生产MucA蛋白， 使用的启动子。 在体外裂解是相当快的 与功能上同源的UmuD相比。 为了研究 在参与诱变DNA修复的蛋白质之间，我们使用了UmuD '蛋白质， 亲和柱。 使用专门的转导λ噬菌体，umuDC 操纵子被删除并被cat基因取代。 的 delta（umuDC）595：：cat突变随后被转移到一个变种中。 基因背景。 我们发现UmuDC蛋白，通常 诱导性诱变所需的，不是细胞存活所必需的。 哺乳动物细胞中DNA损伤修复的过程， 用各种体内或体外系统进行研究。 在灵长类细胞中， 紫外线暴露，DNA修复功能在早期被抑制（由于 修复因子的消耗），然后增强（由于从头合成 这些因素）。 这种修复的调节与 一种新的损伤特异性DNA结合蛋白（DDB）。 DDB可能会 在修复紫外线损伤的DNA的基础上， 体外对紫外线损伤DNA的高亲和力， 细胞在体内，和缺乏在一些患者与紫外线修复缺陷， 着色性干皮病（E组）。 DDB可能是一个家庭的原型， 哺乳动物切除修复蛋白。 SV40蛋白的功能 小T抗原（标签）还没有被很好地理解。 据了解，标签是 所需的有效的大T抗原介导的诱导细胞 当非分裂细胞被感染时， 体外SV40标记，但在增殖细胞中不需要标记。 我们也 发现缺乏标签的SV40突变体能迅速转化增殖的细胞， 型，但不具有低增殖率的细胞。 我们现在已经 发现了tag的生物化学功能：其抑制SV40 DNA的能力 在体外系统中复制。 标签蛋白，通过抑制 细胞磷酸酶PP2A可能改变大T细胞的磷酸化状态， 病毒复制起始所需的抗原。 我们现在搞的 标签也可能抑制抗癌基因活性，解释了 它在非分裂细胞中的需求。

Studies on mutagenesis focus on the UmuDC proteins in E. coli or homologues (e.g., MucAB) in plasmids. A chemiluminescent assay has been used to analyze the processing of UmuD to a mutagenically active form (UmuD'). In E. coli, this process is inefficient, requiring derepressed levels of activated RecA for UmuD cleavage. We have detected a UmuD-like protein in diverse enterobacteria. Several non-mutable strains were defective in UmuD processing, while the poorly mutable S. typhimurium, was, surprisingly, most efficient. To study MucA cleavage, we overproduced the MucA protein using the lambdaPL promoter. In vitro cleavage was substantially faster than that of the functionally homologous UmuD. To study the interactions between proteins involved in mutagenic DNA repair, we used a UmuD' protein affinity column. Using a specialized tranducing lambda phage, the umuDC operon was deleted and replaced with the cat gene. The delta(umuDC)595::cat mutation was subsequently transferred into a variety of genetic backgrounds. We found that the UmuDC proteins, normally required for inducible mutagenesis, are not essential for cell survival. The processes by which DNA lesions are repaired in mammalian cells were studied with various in vivo or in vitro systems. In primate cells, after UV exposure, DNA repair functions are inhibited at early times (due to consumption of repair factors) and then enhanced (due to de novo synthesis of these factors). This modulation of repair is correlated with the level of a novel damage-specific DNA-binding (DDB) protein. DDB is likely to have an important role in the repair of UV-damaged DNA on the basis of its high affinity for UV-damaged DNA in vitro, enhancement by UV exposure of cells in vivo, and absence in some patients with the UV-repair deficiency, xeroderma pigmentosum (group E). DDB may be the prototype of a family of mammalian excision repair proteins. The function of the SV40 protein small t antigen (tag) is not well understood. It is known that tag is required for the efficient large T antigen-mediated induction of cellular DNA replication and transformation when nondividing cells are infected in vitro by SV40, but tag is not required in proliferating cells. We also find that SV40 mutants lacking tag transform rapidly proliferating cell types in vivo, but not cells with a low proliferative rate. We have now found a biochemical function for tag: its ability to inhibit SV40 DNA replication in an in vitro system. The tag protein, by inhibiting the cellular phosphatase PP2A, may alter the phosphorylation state of large T antigen required for the initiation of viral replication. We are pursuing the possibility that tag also inhibits antioncogene activity, explaining its requirement in non-dividing cells.