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

在真核细胞中，基于SV40的穿梭载体pZ189已经被 利用体内和体外，分析突变对紫外线和 苯并芘(BP)。这两种药物产生不同的频率和 体内突变的特异性。此外，突变固定(与 与体内相似的光谱)发生在UV和BP受损的复制过程中 PZ189体外培养(用细胞提取液加SV40大T抗原促进 DNA复制)。在缺口填充突变试验中，纯化的聚合酶 阿尔法(HeLa细胞)在DNA合成过程中高度不准确，但准确性 如果HeLa单链DNA结合蛋白(SSB) 也是存在的。两个BP对映体(+)和(-)anti与 DNA，但(+)形式的致癌性要强得多。当pZI89被引用时 有了两个对映体，载体在体内“穿梭”，(+)形式 在损伤方面对复制复合体有更大的影响 旁路和核苷酸错误掺入。在体外复制系统中， (+)形式再次产生了更大的影响。在大肠杆菌中的研究主要集中在 UmuD切割反应，这在体外是无效的，但需要 用于体内的诱变。我们现在发现，使用一种超敏感的 化学发光分析表明，UmuD在体内的切割效率也很低。至 阐明重要但低效产生的卵裂的作用 产品，UmuD‘，我们通过放置一个 在强启动子lambda PL的控制下，构建了UmuD‘基因。 与大肠杆菌不同，鼠伤寒沙门氏菌的变异性很差。然而，我们发现， 沙门氏菌UmuD蛋白被切割到UmuD‘，表明在 突变不是由于加工过程中的缺陷造成的。当哺乳动物细胞 用致癌物(紫外线或丝裂霉素C)预处理后，紫外线损伤的修复 DNA(以转基因载体的形式)被增强。我们已经确定了一个 构成DNA损伤特异的DNA结合(DDB)蛋白复合体 通过这种预处理被诱导到很高的水平。DDB蛋白是第一个 这种损伤特异的、可诱导损伤的DNA结合蛋白在 灵长类细胞。在慢性阻塞性肺疾病患者中不能检测到或诱导出DDB蛋白 本病修复不足，易患癌症，色素性干皮病。因此， DDB可能在DNA修复中起核心作用。关于DNA复制， 我们的重点是一种病毒基因产物，SV40小T抗原(Tag)。这 蛋白质是有效诱导细胞DNA复制所必需的 和未分裂细胞体外感染SV40时的转化， 而大标签本身就足以增殖细胞。我们发现SV40 标签缺失突变体很容易改变快速增殖的细胞类型 (例如，淋巴母细胞、成骨细胞)，导致淋巴瘤和 感染仓鼠的骨肉瘤；感染野生型SV40的动物 发生非增殖性细胞肿瘤(即纤维肉瘤)。SV40标签 可能影响TAG的磷酸化状态的改变，进而可能 是标签诱导的DNA复制所必需的。

In eukaryotic cells, the SV40-based shuttle vector, pZ189, has been utilized in vivo and in vitro, to analyze mutations in response to UV and benzpyrene (BP). These two agents yield different frequencies and specificities of mutations in vivo. Moreover, mutation fixation (with similar spectra as in vivo) occurs during replication of UV and BP-damaged pZ189 in vitro (using cell extracts plus SV40 large T antigen to promote DNA replication). In a gap-filling mutagenesis assay, purified polymerase alpha (HeLa cell) is highly inaccurate during DNA synthesis, but accuracy is improved several-fold if HeLa single-stranded DNA binding protein (SSB) is also present. Two BP enantiomers, (+) and (-)anti, form adducts with DNA, but the (+) form is far more carcinogenic. When pZI89 was adducted with the two enantiomers, and the vector "shuttled" in vivo, the (+) form had a greater effect on the replication complex both in terms of lesion bypass and nucleotide misincorporation. In the in vitro replication system, the (+) form again had a greater effect. Studies in E.coli have focused on the UmuD cleavage reaction, which is inefficient in vitro but is required for mutagenesis in vivo. We now find, using an ultrasensitive chemiluminescent assay, that UmuD cleavage is also inefficient in vivo. To elucidate the role of the vital but inefficiently produced cleavage product, UmuD', we have over-produced UmuD' protein by placing an engineered UmuD' gene under the control of the strong lambda PL promoter. Unlike E.coli, S.typhimurium is poorly mutable. However, we found that the Salmonella UmuD protein is cleaved to UmuD', indicating that the defect in mutagenesis is not due to a defect in processing. When mammalian cells are pretreated with a carcinogen (UV or mitomycin C), the repair of UV-damaged DNA (in the form of a transfected vector) is enhanced. We have identified a constitutive DNA damage-specific, DNA binding (DDB) protein complex which is induced to high levels by this pretreatment. DDB protein is the first such damage-specific, damage-inducible DNA binding protein identified in primate cells. DDB protein is not detectable nor inducible in patients with the repair deficient, cancer-prone disease, xeroderma pigmentosum. Thus, DDB may play a central role in DNA repair. With regard to DNA replication, our focus is on a viral gene product, SV40 small t antigen (tag). This protein is required for the efficient induction of cellular DNA replication and transformation when non-dividing cells are infected in vitro by SV40, while large Tag alone suffices in proliferating cells. We found that SV40 tag deletion mutants readily transform rapidly proliferating cell types (e.g., lymphoblast, osteoblast) in vivo, leading to lymphomas and osteosarcomas in infected hamsters; animals infected with wild type SV40 develop tumors of nonproliferating cells (i.e., fibrosarcomas). SV40 tag may effect a change in the phosphorylation state of Tag, which may in turn be necessary for Tag-induced DNA replication.