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

原核细胞诱变机制的研究已成为热点 RecA和UmuDC样致突变蛋白的作用 促进DNA聚合酶绕过未修复的DNA损伤)。我们有 大量生产、纯化和结晶了E.coliUmuD‘蛋白。这个 结构被细化到2.5埃，分析表明在 除了与自身形成分子二聚体外，它的氨基末端 UmuD‘可以与另一个“分子二聚体”的氨基末端相互作用 形成延伸的聚合物(“长丝”)结构。在删除时 UmuD‘的N-末端仍然允许蛋白质形成“分子 二聚体“，它阻止聚合物的形成。N-末端 UmuD‘的缺失突变体显示其结合能力大大降低 并解释了为什么表达RecA核蛋白细丝的细胞 突变体(连同UMUC)是不可变的。在其他研究中， 构建了UMUC嵌合蛋白，并对其进行了研究 与鼠伤寒沙门氏菌相关的表型变异较差。这些构造 揭示了位于鼠伤寒沙门氏菌之间的区域的变化 UMUC残基26-59很可能是突变能力较差的原因 表型。我们还考察了UMU的体内稳定性。 大肠杆菌中的蛋白质。UMUC似乎天生就不稳定，但实际上 在Umud的存在下部分稳定下来。具有诱变活性的 UMUD似乎进一步稳定了UMUC；此外，UMUC稳定了 当RecA蛋白在其结构上表达时 激活状态。对非洲爪哇的研究表明，虽然 卵母细胞可以有效地复制未受损的单链DNA，它们是 无法复制包含加合物的DNA。这一复制停止是 在黄体酮成熟卵母细胞和显微注射卵母细胞中减轻 用编码原核UmuD‘C或粘蛋白A’B的mRNAs诱变 蛋白质，这表明诱变的基本机制是高度 在原核细胞和真核细胞之间保守。 在第二个项目中，我们继续研究一种蛋白质(UV-DDB) 在紫外线损伤的人类DNA中与6/4的光产物结合。纯化的UV-DDB 在体外核苷酸切除修复系统中不是必不可少的，但它 在体内可能是必需的。UV-DDB与 紫外线对细胞造成的DNA损伤；RPA，它也可以重新分布 在UV之后，UV-DDB/DNA复合体中也存在。的相互作用 DDB和RPA单独增强了两者的DNA结合。这种互动， 与XPA+RPA类似，将UV-DDB放在初始损害识别中 DNA修复的步骤。

Studies on the mechanism of mutagenesis in prokaryotic cells have focused on the roles of the RecA and UmuDC-like mutagenesis proteins (which facilitate DNA polymerase bypass of unrepaired DNA lesions). We have overproduced, purified and crystallized the E.coli UmuD' protein. The structure was refined to 2.5 angstroms and analyses revealed that in addition to forming a molecular dimer with itself, the amino terminal of UmuD' can interact with the amino terminal of another "molecular dimer" to form an extended polymer ("filament") structure. While deletion of the N-terminal of UmuD' still allows the protein to form the "molecular dimer", it precludes the formation of the polymer. The N-terminal deletion mutant of UmuD' demonstrates a greatly reduced ability to bind to the RecA nucleoprotein filament and explains why cells expressing the mutant (together with UmuC) are non-mutable. In other studies, construction of chimeric UmuC proteins were generated to investigate the poorly mutable phenotype associated with S.typhimurium. These constructs revealed that alterations in the region located between S.typhimurium UmuC residues 26-59 are most likely the cause of the poorly mutable phenotype. We have also investigated the in vivo stability of the Umu proteins in E. coli. UmuC appears to be inherently unstable, but is partially stabilized in the presence of UmuD. The mutagenically active UmuD' appears to stabilize UmuC further; moreover, UmuC was stabilized still further when RecA protein was constitutively expressed in its activated state. Studies with Xenopus laevis demonstrated that while oocytes can efficiently replicate undamaged single-stranded DNA, they are unable to replicate DNA containing adducts. This replication arrest was alleviated in progesterone-matured oocytes and in oocytes microinjected with mRNAs encoding the prokaryotic UmuD'C or MucA'B mutagenesis proteins, suggesting that the basic mechanisms of mutagenesis are highly conserved between prokaryotic and eukaryotic cells. In a second project, we continued studies on a protein (UV-DDB) which binds to 6/4 photoproducts in UV-damaged human DNA. Purified UV-DDB was not essential in an in vitro nucleotide excision repair system, but it may be required in vivo. UV-DDB 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. The interaction of DDB and RPA enhances the DNA binding of either alone. This interaction, similar to XPA + RPA, places UV-DDB in the initial damage recognition step of DNA repair.