Tang M, Bruck I, Eritja R, Turner J, Frank E G, Woodgate R, O'Donnell M, Goodman M F
Department of Biological Sciences, Hedco Molecular Biology Laboratories, University of Southern California, Los Angeles, CA 90089-1340, USA.
Proc Natl Acad Sci U S A. 1998 Aug 18;95(17):9755-60. doi: 10.1073/pnas.95.17.9755.
Damage-induced SOS mutagenesis requiring the UmuD'C proteins occurs as part of the cells' global response to DNA damage. In vitro studies on the biochemical basis of SOS mutagenesis have been hampered by difficulties in obtaining biologically active UmuC protein, which, when overproduced, is insoluble in aqueous solution. We have circumvented this problem by purifying the UmuD'2C complex in soluble form and have used it to reconstitute an SOS lesion bypass system in vitro. Stimulated bypass of a site-directed model abasic lesion occurs in the presence of UmuD'2C, activated RecA protein (RecA*), beta-sliding clamp, gamma-clamp loading complex, single-stranded binding protein (SSB), and either DNA polymerases III or II. Synthesis in the presence of UmuD'2C is nonprocessive on damaged and undamaged DNA. No lesion bypass is observed when wild-type RecA is replaced with RecA1730, a mutant that is specifically defective for Umu-dependent mutagenesis. Perhaps the most noteworthy property of UmuD'2C resides in its ability to stimulate both nucleotide misincorporation and mismatch extension at aberrant and normal template sites. These observations provide a biochemical basis for the role of the Umu complex in SOS-targeted and SOS-untargeted mutagenesis.
需要UmuD'C蛋白的损伤诱导型SOS诱变是细胞对DNA损伤的整体反应的一部分。由于难以获得具有生物活性的UmuC蛋白，体外对SOS诱变的生化基础的研究受到了阻碍，该蛋白过量表达时在水溶液中不溶。我们通过以可溶形式纯化UmuD'2C复合物解决了这个问题，并使用它在体外重建了一个SOS损伤旁路系统。在UmuD'2C、活化的RecA蛋白（RecA*）、β滑动夹、γ夹加载复合物、单链结合蛋白（SSB）以及DNA聚合酶III或II存在的情况下，发生了对位点定向模型无碱基损伤的刺激旁路。在UmuD'2C存在下的合成在受损和未受损的DNA上都是非连续的。当野生型RecA被RecA1730取代时，未观察到损伤旁路，RecA1730是一种对Umu依赖性诱变特别有缺陷的突变体。也许UmuD'2C最值得注意的特性在于其能够在异常和正常模板位点刺激核苷酸错配掺入和错配延伸。这些观察结果为Umu复合物在SOS靶向和SOS非靶向诱变中的作用提供了生化基础。
