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

原核细胞诱变机制的研究主要集中在 RecA和UmuDC样诱变蛋白。我们结晶了UmuD' 蛋白质;将结构细化到2.5埃;并阐明了 自切割机制，将UmuD转化为致突变的 活跃的UmuD '。我们还发现，当UmuD'形成一个分子时， 同二聚体，它也可以形成延伸聚合物结构， 与RecA* 以及UmuC相互作用，促进跨损伤DNA 在未修复的DNA损伤部位合成。我们发现UmuD和 UmuC蛋白在大肠杆菌中本质上不稳定。大肠杆菌，并被降解， 离子蛋白酶。然而，当与UmuD '共表达时，UmuC受到保护。 虽然同二聚体UmuD'蛋白相当稳定，但其快速降解。 当与UmuD形成异二聚体复合物时，被ClpXP蛋白酶降解。 这些相互作用允许易错的，Umu介导的合成仅作为一种 最后一招，避免无端突变。关于X.laevis的研究 证明了卵母细胞可以有效地复制未受损的ss， 它们不能复制含有加合物的DNA。这次逮捕 在孕酮成熟的卵母细胞和 微注射umuDC mRNA，表明其基本机制 诱变是高度保守的。我们分析了 recA 730 lexA 51（Def）菌株的突变子表型 携带一组突变lacZ基因的F_-质粒， 检测特定的转换、颠换和移码事件。最 的自发突变可以归因于特定的增加 在A：T到T：A、A：T到C：G和G：C到T：A颠换中。这些事件 在Δ umuDC菌株中完全消除，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 DNA聚合酶III的3 ′-5 ′校正亚基的缺陷， 这增加了野生型β 2蛋白失活的可能性 在表达RecA 730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载体进行分析 一组等基因E.大肠杆菌Δ umuDC 携带表达UmuD ′ C或其 同源物（mucA ′ B，rumA ′ B）。在umuD 'C菌株中，3' T至A突变 在数量上超过了3' T对C，但对于同源物来说，情况正好相反， 这表明UmuD 'C及其同系物在其相对的 促进从错配末端延伸的能力。迄今为止， 与E.大肠杆菌UmuC蛋白已经被 所有这些似乎都在损伤诱导中发挥关键作用， 肠杆菌科的诱变。远亲UmuC同源物， DinB在E.大肠杆菌，作为一个新成员， 硫磺硫化叶菌[Sulfolobus solfataricus [dbh（dinB 同源物）]。 在第二个项目中，我们研究了蛋白质复合物（UV-DDB/XPE因子） 它与紫外线损伤的人类DNA中的6/4光产物结合。我们发现，在 一种体外核苷酸切除修复（NER）测定， 该复合物导致适度的修复刺激，这表明， UV-DDB在NER过程中起辅助作用，但不是核心作用。我们 还发现这种复合物与受损的DNA紧密结合， 在细胞的UV处理之后。RPA在紫外线照射后也会重新分布， 也存在于UV-DDB/DNA复合物中，DDB和 RPA增强了两者单独的DNA结合，表明UV-DDB 在DNA修复的初始识别步骤中起作用。

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.