MECHANISMS OF INDUCIBLE ERROR PRONE DNA REPLICATION

点击翻译按钮获取中文摘要

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.