UMUD MUTAGENESIS PROTEIN OF ESCHERICHIA-COLI - OVERPRODUCTION, PURIFICATION, AND CLEAVAGE BY RECA
UMUD MUTAGENESIS PROTEIN OF ESCHERICHIA-COLI - OVERPRODUCTION, PURIFICATION, AND CLEAVAGE BY RECA
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DOI：
10.1073/pnas.85.6.1811
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发表时间：
1988-03-01
影响因子：
11.1
通讯作者：
ECHOLS, H
中科院分区：
文献类型：
--
作者：
BURCKHARDT, SE;WOODGATE, R;ECHOLS, H
大肠杆菌的突变率在用复制抑制剂如紫外线处理后增加约100倍。这种增强的突变率需要UmuD和UmuC蛋白的作用，这是作为DNA损伤SOS反应的一部分而诱导的。为了启动这些蛋白质的作用的生化表征，我们已经开发了一种质粒系统，该系统提供umuD和umuC基因的有效表达。将umuD和umuC基因置于受调节的噬菌体的控制下。PL启动子和合成的核糖体结合位点，并调整到UmuD起始的距离以使基因表达最大化。从这个过量生产系统出发，我们纯化了UmuD蛋白，并研究了它与RecA的相互作用。通过RecA蛋白介导莱克萨阻遏物切割SOS控制的操纵子的能力，开启SOS应答。其他人已经表明，UmuD在切割位点周围表现出与莱克萨的序列同源性，这表明UmuD可能存在切割反应。我们发现，RecA介导的切割UmuD，可能在这个网站。与莱克萨一样，UmuD也经历自裂解反应。我们推断RecA介导的UmuD切割是RecA在SOS诱变中的另一个作用，可能激活UmuD的诱变功能。
The mutation rate of Escherichia coli increases approximately 100-fold after treatment with replication-inhibiting agents such as UV light. This enhanced mutation rate requires the action of the UmuD and UmuC proteins, which are induced as part of the SOS response to DNA damage. To initiate a biochemical characterization of the role of these proteins, we have developed a plasmid system that gives efficient expression of the umuD and umuC genes. The umuD and umuC genes were placed under the control of a regulated phage .lambda. PL promoter and a synthetic ribosome-binding site, and the distance to the UmuD start was adjusted to maximize gene expression. Starting from this overproduction system, we have purified the UmuD protein and studied its interaction with RecA. The SOS response is turned on by the capacity of RecA protein to mediate cleavage of the LexA repressor for SOS-controlled operons. Others have shown that UmuD exhibits sequence homology to LexA around the cleavage site, suggesting a possible cleavage reaction of UmuD. We show that RecA mediates cleavage of UmuD, probably at this site. As with LexA, UmuD also undergoes a self-cleavage reaction. We infer that RecA-mediated cleage of UmuD is another role for RecA in SOS mutagenesis, probably activating UmuD for its mutagenic function.