MECHANISMS OF INDUCIBLE ERROR PRONE DNA REPLICATION

诱变剂通过两条不同的途径起作用：第一条是通过 由诱变剂造成的DNA损伤的直接错误复制。第二 是诱变剂改变细胞的间接途径 生理学方面的增强诱变作用不仅限于同源DNA 损伤，但也存在于其他(异源)DNA损伤 基因组，以及未受损的细胞。大肠埃希菌SOS现象 代表了最广为人知的诱变途径。我们最近做了 描述了UVM，一种非SOS依赖的DNA损伤诱导的突变反应 在大肠杆菌中。这项提议中的假设是DNA损伤 在大肠杆菌中诱导了两种平行的现象，即SOS和UVM 在可诱导突变中的非重叠作用。UVM建议用于 在非编码病变处调制碱基插入(UV调制 突变)，而SOS反应则绕过停滞的位置 复制。1类DNA损伤，如基本部位，需要SOS 旁路步骤的功能，而2类病变(3，N4- 乙烯胞嘧啶，epsilonC)没有。对这一假设的两种预测 将使用高分辨率实验系统进行测试，在该系统中，M13 携带特定位点DNA的病毒单链DNA分子 损伤将被用来探测突变固定环境 手机。体内和体外病变的突变将通过以下方法进行分析 一种不需要基于表型选择的测序技术 或者筛选变种人。来测试以下预测： 诱导水平的SOS蛋白是必要的，但不足以 解释了在紫外线照射下观察到的容易出错的复制 细胞，具有recA、umuD和recuD等位基因的大肠杆菌菌株 UMUC基因被置于可调控的异源基因控制之下 发起人将被构建。突变固定的特征将是 在1类和2类病变中，未诱导和UVM诱导的细胞 SOS蛋白的表达不受DNA损伤的影响。DNA是否 UVM诱导的细胞的复制被改变将通过寻找 在无细胞提取物中存在容易出错的复制能力 从UVM诱导的细胞中制备。UVm构成的大肠杆菌突变体 最近被隔离将被描述为朝着隔离的第一步 UVM-缺陷细胞。

Mutagens act through two distinct pathways: the first is through the direct misreplication of DNA damage inflicted by the mutagen. The second is an indirect pathway in which the mutagens alter the cellular physiology such as to enhance mutagenesis not only at the cognate DNA lesions, but also at other (heterologous) DNA damage present in the genome, and at undamaged cells. The Escherichia coli SOS phenomenon represents the best known inducible mutagenic pathway. We have recently described UVM, an SOS-independent DNA damage-inducible mutagenic response in Escherichia coli. The hypothesis in this proposal is that DNA damage in E. coli induces two parallel phenomena, namely, SOS and UvM, that play non-overlapping roles in inducible mutagenesis. UVM is proposed to modulate base insertion at noncoding lesions (UV modulation of mutagenesis), whereas the SOS response acts to bypass sites of stalled replication. Class 1 DNA lesions such as abasic sites require SOS functions for the bypass step, whereas Class 2 lesions (3,N4- ethenocytosine, epsilonC) do not. Two predictions of this hypothesis will be tested using a high resolution experimental system in which M13 viral single-stranded DNA molecules bearing defined site-specific DNA lesions will be used to probe the mutation fixation environment of the cell. Mutagenesis at the lesion in vivo and in vitro will be analyzed by a sequencing technology that does not require phenotype-based selection or screening for mutants. To test the prediction that the expression of SOS proteins at induced levels will be necessary but not sufficient to account for inducible error-prone replication observed in UV-irradiated cells, E. coli strains in which appropriate alleles of the recA, umuD and umuC genes are placed under the control of regulatable heterologous promoters will be constructed. Mutation fixation will be characterized at Class 1 and Class 2 lesions uninduced and UVM-induced cells in which the expression of SOS proteins is uncoupled from DNA damage. Whether DNA replication is altered in UVM induced cells will be tested by seeking the presence of error-prone replication capability in cell-free extracts prepared from UVM-induced cells. UVM-constitutive E. coli mutants recently isolated will be characterized as a first step toward isolating UVM-defective cells.