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

原核细胞诱变机制研究的重点 RecA和UmuDC样致突变蛋白。我们使UMUD具体化了 蛋白质；将结构细化到2.5埃；并阐明了 将UmuD转化为诱变性UmuD的自我切割机制 活跃的Umud‘。我们还发现，当UmuD‘形成一个分子时 同二聚体，它还可以形成延伸的聚合物结构， 与RecA*和UMUC相互作用，促进跨损伤DNA 未修复的DNA损伤部位的合成。我们发现UMUD和 UMUC蛋白在大肠杆菌中天生不稳定，并被 隆蛋白水解酶。然而，当UMUC与UmuD‘共表达时，UMUC受到保护。 虽然同源二聚体UmuD‘蛋白相当稳定，但它很快 在与UmuD形成异二聚体复合体时被ClpXP蛋白酶降解。 这些交互只允许容易出错的UMU中介的合成作为 最后的手段，避免无端的突变。与X.laevis的研究 证明了虽然卵母细胞可以有效地复制未受损的SS DNA，它们不能复制含有加合物的DNA。这次逮捕 在黄体酮成熟卵母细胞和卵母细胞中的表达均有所减轻 显微注射umuDC mRNAs，提示其基本机制 突变是高度保守的。我们分析了这种病毒的特异性 一株recA730 lexA51(Def)菌株的诱变子表型 携带一组突变的LacZ基因的F_-质粒，这些基因可以单独 检测特定的过渡、转换和移码事件。多数 的自发性突变可以归因于特定的增加 在A：T到T：A中，A：T到C：G和G：C到T：A互换。这些事件是 在Delta umuDC株中完全消失，表明 具有功能活性的UmuD‘C蛋白通常是其 一代。所获得的光谱与具有AFP的菌株相似 DNA聚合酶III的epsilon(3‘-5’校对)亚基缺陷， 增加了野生型epsilon蛋白失活的可能性 在表达RecA730和UmuD‘C蛋白的菌株中。我们还使用了 携带位点特异性T-T环丁烷二聚体的SS DNA载体分析 一组同基因大肠杆菌Delta umuDC的诱变特异性 含有低拷贝数表达UmuD‘C或UmuD’C基因的菌株 同系物(粘蛋白A‘B、鲁马B)。在umuD‘C株中，3’T到A突变 数量超过了3‘T比C，但同系物的情况正好相反， 这表明UmuD‘C及其同系物可能在相对位置上有所不同 从不匹配的末端促进伸长的能力。到目前为止，有8个 与大肠杆菌UMUC蛋白密切相关的同源物已经被 已确定，所有这些似乎都在可诱发损害中发挥关键作用 肠杆菌科中的诱变作用。一个远亲的UMUC同源基因， 在大肠杆菌中也发现了DinB，这是一个新的成员 UMUC-始祖亚目中的超家族 Homolog)]。 在第二个项目中，我们研究了一种蛋白质复合体(UV-DDB/XPE因子) 它与紫外线损伤的人类DNA中6/4的光产物结合。我们发现，在 一种体外核苷酸切除修复(NER)试验，即添加 该复合体导致适度的修复刺激，表明 UV-DDB在NER过程中起辅助作用，但不是核心作用。我们 还发现，该复合体与受损的DNA密切相关 对细胞进行紫外线处理。RPA在紫外线照射后也会重新分发， 也存在于UV-DDB/DNA复合体中，以及DDB与DNA的相互作用 RPA单独增强了两者的DNA结合，表明UV-DDB 在DNA修复的初始识别步骤中发挥作用。

Studies on the mechanism of mutagenesis in prokaryotic cells focused on RecA and UmuDC-like mutagenesis proteins. We crystallized the UmuD' protein; refined the structure to 2.5 angstroms; and elucidated the mechanism of self-cleavage which converts UmuD to the mutagenically active UmuD'. We also discovered that while UmuD' forms a molecular homodimer, it can also form an extended polymeric structure which interacts with RecA* as well as UmuC, facilitating translesion DNA synthesis at the site of unrepaired DNA damage. We found that UmuD and UmuC proteins are inherently unstable in E. coli and are degraded by the Lon protease. UmuC is, however, protected when co-expressed with UmuD'. While the homodimeric UmuD' protein is quite stable, it is rapidly degraded by the ClpXP protease when in a heterodimeric complex with UmuD. These interactions allow error-prone, Umu-mediated synthesis only as a last resort, avoiding gratuitous mutagenesis. Studies with X.laevis demonstrated that while oocytes can efficiently replicate undamaged ss DNA, they are unable to replicate DNA which contains adducts. This arrest was alleviated in progesterone-matured oocytes and in oocytes microinjected with umuDC mRNAs, suggesting that the basic mechanisms of mutagenesis are highly conserved. We analyzed the specificity of the mutator phenotype of a recA730 lexA51(Def) strain by employing F_-plasmids carrying a set of mutant lacZ genes that can individually detect specific transitions, transversions, and frameshift events. Most of the spontaneous mutagenesis could be attributed to a specific increase in A:T to T:A, A:T to C:G and G:C to T:A transversions. These events were completely abolished in a delta umuDC strain, indicating that functionally active UmuD'C proteins are normally required for their generation. The spectrum obtained was similar to that of strains with a defect in the epsilon (3'-5' proofreading) subunit of DNA polymerase III, raising the possibility that the wild-type epsilon protein is inactivated in strains expressing the RecA730 and UmuD'C proteins. We also used an ss DNA vector carrying a site-specific T-T cyclobutane dimer to analyze the mutagenic specificity of a set of isogenic E. coli delta umuDC strains harboring low-copy-number plasmids expressing UmuD'C or its homologs (mucA'B, rumA'B). In umuD'C strains, 3' T to A mutations outnumbered 3' T to C, but the reverse was true for the homologs, suggesting that UmuD'C and its homologs may differ in their relative abilities to promote elongation from mismatched termini. To date, eight closely related homologs of the E. coli UmuC protein have been identified, with all appearing to play critical roles in damage-inducible mutagenesis in enterobacteriaceae. A distantly related UmuC-homolog, DinB, was also identified in E. coli, as was a new member of the UmuC-superfamily in the archeon, Sulfolobus solfataricus [dbh (dinB homolog)]. In a second project, we studied a protein complex (UV-DDB/XPE factor) which binds to 6/4 photoproducts in UV-damaged human DNA. We found, in an in vitro nucleotide excision repair (NER) assay, that the addition of the complex leads to a modest stimulation of repair, suggesting that UV-DDB plays an accessory, but not a core role, in the NER process. We also found that the complex moves to a tight association with damaged DNA upon UV treatment of cells. RPA, which also redistributes after UV, is also present in the UV-DDB/DNA complex, and the interaction of DDB and RPA enhances the DNA binding of either alone, suggesting that UV-DDB functions in the initial recognition step of DNA repair.