MECHANISMS OF INDUCIBLE ERROR PRONE DNA REPLICATION

诱变剂通过两种不同的途径起作用：第一种是通过 诱变剂造成的DNA损伤的直接错误复制。第二 是一种间接途径，其中诱变剂改变细胞 例如不仅在同源DNA处增强诱变 损伤，但也在其他（异源）DNA损伤存在于 基因组和未受损的细胞。大肠杆菌SOS现象 代表了已知最好的诱导性诱变途径。 我们最近 描述了UVM，一种不依赖SOS的DNA损伤诱导的诱变反应， 在大肠杆菌中。 该提案中的假设是DNA损伤 在大肠大肠杆菌诱导两个平行的现象，即SOS和UvM，发挥 在诱导突变中的非重叠作用。 建议UVM 调节非编码损伤处的碱基插入（UV调节 突变），而SOS反应的行为，以绕过网站的停滞 复制的1类DNA病变，如脱碱基位点，需要SOS 功能为旁路步骤，而2类病变（3，N4- 乙烯胞嘧啶，ε C）不。 这个假设的两个预测 将使用高分辨率实验系统进行测试，其中M13 带有确定的位点特异性DNA的病毒单链DNA分子 病变将被用来探测突变的固定环境， cell.将通过以下方法分析体内和体外病变处的突变： 不需要基于表型选择的测序技术 或者筛选突变体为了验证预测， 诱导水平的SOS蛋白将是必要的，但不足以 解释了在紫外线照射的细胞中观察到的可诱导的易错复制 cells，E.其中recA、umuD和 将umuC基因置于可调控的异源性 发起人将组建。 突变固定将被表征 在1类和2类病变中，未诱导和UVM诱导的细胞，其中 SOS蛋白的表达与DNA损伤解偶联。无论是DNA 复制在UVM诱导的细胞中的改变将通过寻找 无细胞提取物中存在易错复制能力 由紫外线诱导的细胞制备。紫外线组成型E.大肠杆菌突变株 最近被隔离的人将被描述为隔离的第一步 紫外线缺陷细胞。

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.