Peat T S, Frank E G, McDonald J P, Levine A S, Woodgate R, Hendrickson W A
Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032, USA.
Structure. 1996 Dec 15;4(12):1401-12. doi: 10.1016/s0969-2126(96)00148-7.
Damage induced 'SOS mutagenesis' may occur transiently as part of the global SOS response to DNA damage in bacteria. A key participant in this process is the UmuD protein, which is produced in an inactive from but converted to the active form, UmuD', by a RecA-mediated self-cleavage reaction. UmuD', together with UmuC and activated RecA (RecA*), enables the DNA polymerase III holoenzyme to replicate across chemical and UV induced lesions. The efficiency of this reaction depends on several intricate protein-protein interactions.
Recent X-ray crystallographic analysis shows that in addition to forming molecular dimers, the N- and C-terminal tails of UmuD' extend from a globular beta structure to associate and produce crystallized filaments. We have investigated this phenomenon and find that these filaments appear to relate to biological activity. Higher order oligomers are found in solution with UmuD', but not with UmuD nor with a mutant of UmuD' lacking the extended N terminus. Deletion of the N terminus of UmuD' does not affect its ability to form molecular dimers but does severely compromise its ability to interact with a RecA-DNA filament and to participate in mutagenesis. Mutations in the C terminus of UmuD' result in both gain and loss of function for mutagenesis.
The activation of UmuD to UmuD' appears to cause a large conformational change in the protein which allows it to form oligomers in solution at physiologically relevant concentrations. Properties of these oligomers are consistent with the filament structures seen in crystals of UmuD'.
损伤诱导的“SOS诱变”可能作为细菌对DNA损伤的全局SOS反应的一部分而短暂发生。这一过程中的一个关键参与者是UmuD蛋白，它以无活性形式产生，但通过RecA介导的自我切割反应转化为活性形式UmuD'。UmuD'与UmuC和活化的RecA（RecA*）一起，使DNA聚合酶III全酶能够跨越化学和紫外线诱导的损伤进行复制。该反应的效率取决于几种复杂的蛋白质-蛋白质相互作用。
最近的X射线晶体学分析表明，除了形成分子二聚体外，UmuD'的N端和C端尾巴从球状β结构延伸出来，相互结合并产生结晶细丝。我们研究了这一现象，发现这些细丝似乎与生物活性有关。在含有UmuD'的溶液中发现了高阶寡聚体，但在含有UmuD的溶液中未发现，在缺乏延伸N端的UmuD'突变体溶液中也未发现。删除UmuD'的N端并不影响其形成分子二聚体的能力，但确实严重损害了其与RecA-DNA细丝相互作用并参与诱变的能力。UmuD' C端的突变导致诱变功能的获得和丧失。
UmuD向UmuD'的激活似乎导致蛋白质发生大的构象变化，使其能够在生理相关浓度下在溶液中形成寡聚体。这些寡聚体的性质与UmuD'晶体中看到的细丝结构一致。
