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

这就是大肠杆菌。UMU操纵子编码两种突变蛋白(UmuD，C) 在复制过程中通过DNA聚合酶促进病变的旁路 DNA受损。发现了UmuD、C的功能同系物，例如，粘蛋白A、B 自然产生的质粒组。我们发现翻译后的 将UmuD蛋白加工成具有诱变活性的形式UmuD‘，是 效率相对较低。高度诱变性的粘液A被加工成 非常高效。UmuD和UmuD‘蛋白与RecA发生物理相互作用 蛋白质，这种相互作用可能提供了一种机制，通过这种机制 蛋白质是针对DNA损伤的。三角洲的建设(UmuDC) 突变显示UmuD，C蛋白，正常情况下需要 可诱导的突变对细胞的生存并不是必需的。细菌 携带缺失的菌株已经被构建成允许 UmuDC样基因的快速克隆。我们还研究了识别 以及移除哺乳动物的DNA损伤。在灵长类细胞中，我们鉴定出， 表征并确定了一种新的核的初级序列 紫外线损伤特异性DNA结合蛋白(UV-DDB)。这种蛋白质， 相对较新的进化起源，可通过以下方式诱导到高水平 紫外线。UV-DDB蛋白可能参与DNA修复的早期步骤 (例如，损伤识别)基于其对紫外线损伤的DNA的高亲和力 在体外，紫外光对它的调节，体内，以及在一些 遗传性紫外线修复缺陷患者色素性干皮病 (E组)。我们已经克隆了编码UV-DDB的基因并进行了测序 蛋白质；该基因的结构缺陷可能是XP-E的原因。 在DNA复制的研究中，我们使用了DNA病毒SV40。角色 在SV40癌基因中，大T抗原(Tag)是众所周知的病毒和 细胞DNA复制。然而，另一种SV40基因产物，小T 抗原(T)，是一个谜。这种蛋白质是标签介导的必需的 诱导细胞DNA复制和转化 未分裂的细胞受SV40感染，但在 正在增殖的细胞。我们已经确定了t的一种生化功能： 它在体外抑制标签介导的病毒DNA复制。我们还发现 TDelta突变体迅速改变了增殖细胞类型(例如， 淋巴细胞)，但不是低增殖率的细胞(例如， 成纤维细胞)。小T可能通过抑制细胞磷酸酶， PP2A，改变其作用所需的TAG的磷酸化状态 在病毒和细胞DNA复制中。标签也已知用于绑定和 灭活几种抑癌基因产物，如Rb、P53和p3OO。这 相互作用也可能受到t的影响。

The E. coli. umu operon encodes two mutagenic proteins (UmuD,C) which facilitate bypass of lesions by DNA polymerase during replication of damaged DNA. Functional homologues of UmuD,C, e.g., MucA,B, are found on naturally occurring plasmids. We found that the post-translational processing of UmuD protein to its mutagenically active form, UmuD', is relatively inefficient. MucA, which is highly mutagenic, is processed very efficiently. UmuD and UmuD'proteins physically interact with RecA protein, and this interaction may provide a mechanism by which the Umu proteins are targeted to DNA lesions. Construction of a delta(umuDC) mutation showed that the Umu D,C proteins, normally required for inducible mutagenesis, are not essential for cell survival. Bacterial strains carrying the deletion have been constructed which permit the rapid cloning of umuDC-like genes. We have also studied the recognition and removal of mammalian DNA lesions. In primate cells, we identified, characterized, and determined the primary sequence of, a novel nuclear UV damage-specific DNA binding (UV-DDB) protein. This protein, of relatively recent evolutionary origin, is inducible to high levels by UV. The UV-DDB protein may be involved in early steps of DNA repair (e.g., lesion recognition) based on its high affinity for UV damaged DNA in vitro, its regulation by UV light in vivo, and its absence in some patients with the heritable UV repair deficiency, xeroderma pigmentosum (group E). We have cloned and sequenced the gene encoding the UV-DDB protein; a structural defect in this gene may be responsible for XP-E. In studies on DNA replication, we utilized the DNA virus, SV40. The role of the SV40 oncogene, large T antigen (Tag) is well known in viral and cellular DNA replication. However, a second SV40 gene product, small t antigen (t), is enigmatic. This protein is required for Tag-mediated induction of cellular DNA replication and transformation when nondividing cells are infected by SV40, but t is not required in proliferating cells. We have identified a biochemical function for t: It inhibits Tag-mediated viral DNA replication in vitro. We also found that tdelta mutants transform rapidly proliferating cell types (e.g., lymphocytes), but not cells with a low proliferative rate (e.g., fibroblasts). Small t may, by inhibiting the cellular phosphatase, PP2A, alter the phosphorylation state of Tag required for its roles both in viral and cellular DNA replication. Tag is also known to bind and inactivate several antioncogene products, e.g., Rb, p53, and p3OO. This interaction may also be affected by t.