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

对原核生物诱变机制的研究主要集中在 RecA和UmuD，C类突变蛋白的作用 在复制过程中通过DNA聚合酶促进病变的旁路 损坏的DNA)。生化分析显示，UmuD，它的裂解 产物(UmuD‘)，以及功能上同源的、质粒编码的 粘蛋白A的蛋白质与RecA物理上相互作用。这种交互可以 提供了一种机制，通过该机制，UMU样蛋白被靶向 DNA中的损伤。已经构建了两个大肠杆菌菌株，使其能够 基于一种简单表型的UMU表型鉴定 互补试验。这些菌株为克隆 来自R-质粒R391、R446b和R471a的三个新的类umu操纵子。 这些测试菌株还被用来鉴定几个新的质粒- 编码的UMUC突变体。在哺乳动物DNA修复的研究中，我们 确定了紫外光对亚细胞的分布和调节 灵长类紫外线损伤DNA结合蛋白的127 kDa组分 DDB)复合体。该UV-DDB蛋白的结构同源物是 在粘菌和大米中鉴定，以及果蝇的部分cdna 同源基因被分离出来。新的证据支持了 哺乳动物DNA损伤识别与修复中的UV-DDB复合体 有缺陷的额外的着色性干皮病患者(“XP变种”) 在UV-DDB中确定了活性(现在已知有4名患者缺乏 这种损害识别活动)；UV-DDB活性的恢复是 XP A、D和C组患者紫外线照射后细胞延迟， 这与它们的DNA修复缺陷有很好的相关性； 通常不表达UV-DDB活性的哺乳动物组织是 比它们的克隆菌株对紫外线更敏感，后者已经恢复了 结合活性。在对SV40作为真核复制子模型的研究中，我们 重点研究了病毒的小T抗原。在体外纯化的SV40 DNA中 复制系统，我们发现小T抑制大T抗原- 从属复制。然而，在体内，Small-t增强了SV40 DNA 复制。感染实验还表明，Small-T刺激 许可猴细胞的进展--但不是非许可啮齿动物 细胞--从细胞周期的G0/G1期到S期，推测 从而为病毒复制提供最佳的细胞内环境。在……里面 啮齿动物实验，小t突变体优先快速转化 增殖的淋巴样细胞，似乎无法转化为 通常有丝分裂率较低，如间皮细胞。在这些 实验中，我们还发现野生型SV40会诱发间皮瘤 在仓鼠身上。随后，我们发现，超过60%的人类 间皮瘤含有和表达SV40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.