ANALYSES OF DISULFIDE LINKED HOMODIMERS OF UMUD & DERIVATIVES

需要SOS调节的UmuDC操纵子的产物用于 大多数诱变是由暴露于紫外线辐射或许多 化学品 当完整的UmuD蛋白与激活的 RecA，它经历了一个促进自身消化，去除24个氨基酸， 从N-末端的氨基酸以产生UmuD '。 这种UmuD的分裂 激活它在诱变中的作用。 我们扩展了对 我们的一组UmuD的生物活性单茎衍生物， 通过构建一组UmuD和UmuD'的单半胱氨酸变体， 其中在位置133、134、135、136 137和138（使用UmuD编号）。 能力分析 这些衍生物在加成后通过二硫键交联 的碘支持NMR实验的结论[Ferentz等人， Nature Structural Biology 4：979-983（1997）]，其C-末端 UmuD ′ 2在溶液中彼此接近。 我们观察到类似的 与UmuD 2的相应单半胱氨酸衍生物交联 同源二聚体表明UmuD ′ 2和UmuD 2同源二聚体界面 都有一个共同的特征，即具有相互作用的C-末端。 然而我们的 位置处的相应半胱氨酸的行为的比较 如UmuD ′ 2与UmuD ′ 2的37、38和57， 分子结构的某些其他特征是非常不同的 UmuD ′ 2和UmuD 2之间。 二硫键连接的 通过质谱法证实了二聚体。 综合这些 结果使我们能够制定一个模型， 发生和裂解后的溶液结构的影响， umuD基因产物（手稿在准备中）。

The products of the SOS-regulated UmuDC operon are required for most of the mutagenesis caused by exposure to UV radiation or many chemicals. When the intact UmuD protein interacts with activated RecA, it undergoes a facilitated autodigestion that removes 24 amino acids from the N-terminus to generate UmuD'. This cleavage of UmuD activates it for its role in mutagenesis. We extended our analyses of our set of biologically active monocysteme derivatives of UmuD and UmuD' by constructing a set of monocysteine variants of UmuD and UmuD' in which cysteines were substituted at positions 133, 134, 135, 136, 137, and 138 (using UmuD numbering). Analyses of the abilities of these derivatives to crosslink by a disulfide bond after the addition of iodine supported the conclusion of NMR experiments [Ferentz et al., Nature Structural Biology 4:979-983 (1997)] that the C-termini of UmuD'2 are near each other in solution. Our observation of similar crosslinking with corresponding monocysteine derivatives of theUmuD2 homodimer suggest that the homodimer interfaces of UmuD'2 and UmuD2 share the common feature of having interacting C-termini. However our comparisons of the behavior of corresponding cysteines at positions such as 37, 38, and 57 of UmuD'2 versus UmuD2 had revealed that certain other features of molecular structure are very different between UmuD'2 and UmuD2. The identities of the disulfide-linked dimers were confirmed by mass spectrometry. Taken together these results have allowed us to formulate a model for how UmuD cleavage occurs and for the effects of cleavage upon the solution structure of the umuD gene product (manuscript in preparation).