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UmuD，C样诱变蛋白（ 在复制过程中通过DNA聚合酶促进病变的旁路， 受损的DNA）。生物化学分析显示，UmuD，其裂解 产物（UmuD '），以及功能上同源的质粒编码的 MucA蛋白质与RecA物理相互作用。这种相互作用可能 提供了一种机制，通过这种机制，Umu样蛋白被靶向于 DNA损伤两个大肠大肠杆菌菌株已经构建， 基于简单表型鉴定Umu表型 互补分析这些菌株有助于克隆 三个新的umu样操纵子来自R质粒R391、R446 b和R471 a。 这些试验菌株还用于鉴定几种新型质粒- 编码umuC突变体。在哺乳动物DNA修复的研究中，我们 通过紫外光测定了 灵长类动物UV损伤的DNA结合（UV- DDB）复合物。这种UV-DDB蛋白的结构同源物是 在黏菌和水稻中鉴定，以及果蝇的部分cDNA 分离同源物。新的证据表明， UV-DDB复合物在哺乳动物DNA损伤识别和修复中的作用 有缺陷的其他着色性干皮病患者（“XP变体”） 在UV-DDB活性被确定（4名患者现在已知缺乏 这种损伤识别活性）; UV-DDB活性的恢复是 在XP A、D和C组患者的UV照射细胞中延迟， 这与它们的DNA修复缺陷密切相关; 通常不表达UV-DDB活性的哺乳动物组织是 比它们的克隆分离物更敏感， 结合活性。在SV 40作为真核生物复制子模型的研究中， 专注于病毒小T抗原 在纯化的体外SV 40 DNA中， 复制系统，我们发现小T抑制大T抗原- 依赖复制然而，在体内，小t增强SV 40 DNA， 复制的感染实验还表明，小t刺激 许可猴细胞进展-但非许可啮齿动物细胞进展 细胞-从G 0/G1到细胞周期的S期，大概 导致病毒复制的最佳细胞内环境。 在 啮齿动物实验中，小t突变体优先迅速转化 增殖的淋巴样细胞，似乎无法转化细胞， 正常情况下有丝分裂率低，如间皮细胞。在这些 实验中，我们还发现野生型SV 40诱导间皮瘤， 在仓鼠身上。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 UmuD, C-like mutagenesis proteins (which facilitate bypass of lesions by DNA polymerase during replication of damaged DNA). Biochemical assays have revealed that UmuD, its cleavage product (UmuD'), and the functionally homologous, plasmid-encoded 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 their DNA repair-deficiency; 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 SV40 DNA replication system, we found that small-t inhibits large T antigen- dependent replication. However, in vivo, small-t enhances SV40 DNA replication. Infection experiments also indicate that small-t stimulates progression of permissive monkey cells - but not 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.