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

原核生物诱变机制的研究主要集中在 RecA和UmuDC样诱变蛋白的作用。生物化学测定 已经揭示了UmuD、UmuD'和功能上同源的MucA' 蛋白质与RecA物理相互作用。这种相互作用可以提供 Umu样蛋白靶向DNA损伤的机制。 两个大肠大肠杆菌菌株已经构建， Umu表型的基础上一个简单的表型互补测定。 这些菌株促进了三个新的umu样操纵子的克隆 来自R质粒R391、R446 b和R471 a。这些试验菌株也 用于鉴定几种新的质粒编码的umuC突变体。在 哺乳动物DNA修复的研究，我们确定了亚细胞 分布和调节的127 kDa的蛋白质组分的紫外光 一种灵长类紫外线损伤DNA结合（UV-DDB）复合物。的结构同系物 这种UV-DDB蛋白在黏菌和水稻中被鉴定， 分离果蝇同源物的cDNA。新的证据表明， UV-DDB复合物在哺乳动物DNA损伤识别中的重要作用 和修复：1名额外的着色性干皮病患者（“XP变体”） 在UV-DDB活性的缺陷被确定（4名患者现在已知 缺乏这种损伤识别活动）; UV-DDB的恢复 在XP组A、D和C的UV照射细胞中，活性延迟 患者，这与DNA修复缺陷密切相关， 这些细胞;以及来自哺乳动物组织的细胞， 表达UV-DDB活性比它们的克隆分离物更UV敏感 其已经恢复了结合活性。在以SV 40为模型的研究中， 真核复制子，我们集中在病毒小T抗原。 中 在纯化的体外DNA复制系统中，我们发现小t抑制了 大T抗原依赖性SV 40 DNA复制。然而，在体内，小t 增强病毒DNA复制。小t突变体的感染实验 病毒表明，这种抗原刺激了允许的 猴细胞-但不是非允许的啮齿动物细胞-从G 0/G1到 细胞周期的S期，可能导致最佳的 病毒复制的细胞内环境。 在啮齿动物实验中， 小t突变体优先转化快速增殖的淋巴样 细胞，似乎无法转化细胞，通常有低 有丝分裂率，如间皮细胞。在这些实验中，我们还 发现野生型SV 40诱导仓鼠间皮瘤。 随后，我们发现超过60%的人类间皮瘤 含有并表达SV 40样序列。

Studies on the mechanism of prokaryotic mutagenesis have focused on the roles of the RecA and UmuDC-like mutagenesis proteins. Biochemical assays have revealed that UmuD, UmuD' and the functionally homologous MucA' proteins physically interact with RecA. This interaction may provide a mechanism by which the Umu-like proteins are targeted to lesions in DNA. Two E. coli strains have been constructed that allow the identification of Umu phenotypes based upon a simple phenotypic complementation assay. These strains have facilitated the cloning of three new umu-like operons from R-plasmids R391,R446b and R471a. These tester strains were also utilized to identify several novel plasmid-encoded umuC mutants. In studies on mammalian DNA repair, we determined the subcellular distribution and regulation by UV light of a 127 kDa protein component of a primate UV-damaged DNA-binding (UV-DDB) complex. Structural homologs of this UV-DDB protein were identified in slime mold and rice, and a partial cDNA of a Drosophila homolog was isolated. New evidence supports an important role for the UV-DDB complex in mammalian DNA damage-recognition and repair: One additional Xeroderma Pigmentosum patient ("XP variant") with a defect in UV-DDB activity was identified (4 patients are now known to lack this damage-recognition activity); the recovery of UV-DDB activity is delayed in UV-irradiated cells from XP groups A, D, and C patients, which correlates well with the DNA repair-deficiency found in these cells; and cells from mammalian tissues that do not normally express UV-DDB activity are more UV sensitive than their clonal isolates which have regained the binding activity. In studies on SV40 as a model eukaryotic replicon, we focused on the viral small t antigen. In a purified in vitro DNA replication system, we found that small t inhibits large T antigen-dependent SV40 DNA replication. However, in vivo, small t enhances viral DNA replication. Infection experiments with small-t mutant viruses indicate that this antigen stimulates progression of permissive monkey cells - but not of non-permissive rodent cells - from the G0/G1 to the S phase of the cell cycle, presumably leading to an optimal intracellular environment for viral replication. In rodent experiments, small-t mutants preferentially transformed rapidly proliferating lymphoid cells and appeared unable to transform cells that normally have a low mitotic rate, such as mesothelial cells. In these experiments, we also found that wild-type SV40 induces mesotheliomas in hamsters. Subsequently, we have found that more than 60% of human mesotheliomas contain and express SV40-like sequences.