Mechanisms of Inducible Error Prone DNA Replication

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DESCRIPTION (provided by applicant): 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 triggering (cognate) DNA lesions, but also at other (heterologous) DNA damage present in the genome, and at undamaged cells. Investigation of such "transient mutator" phenotypes at a fundamental level is necessary for understanding the mechanisms underlying genetic variability in response to environmental conditions, and has impact on healthcare issues such as the paradoxical accrual of multiple mutations in cancerous cells despite normally low mutation rates (10-6/gene/generation), and rapid development of resistance to anti-cancer agents. UVM (for UV modulation of mutagenesis) is a recently described recA-independent DNA damage-inducible mutagenic response in Escherichia coil discovered in our laboratory. UVM promotes mutagenesis at Class 2 noninstructive mutagenic DNA lesions such as 3,N4-ethenocytosine (EC) in an SOS-independent manner, and at Class 1 noninstructive mutagenic lesions such as AP sites in an SOS-dependent manner. The working hypothesis in this proposal is that DNA damage in E. coli triggers two parallel phenomena, namely, SOS and UVM, that play complementary lesion-dependent roles in inducible mutagenesis. The genetic and biochemical basis of the UVM pathway will be pursued in this proposal through two specific aims. Aim 1: We will identify and characterize genes involved in the UVM pathway through the following approaches. (a) Apply a 2-step screen for UVM-defective mutants based on sensitivity to DNA damaging agents, and effect on UVM. (b) Identify most genes whose transcription is altered upon UVM induction by using the Affymetrix GeneChip technology. Aim 2: We will test the hypothesis that UVM is mediated by a transiently altered DNA polymerase III (pol-IlI) through following approaches. (a) Determine the kinetics of IJVM induction in vivo and in vitro. (b) Analyze error-prone replication in UVM-induced dnaE(Ts) mutants (defective for pol-Ill) under permissive and restrictive conditions. (c) Purify and characterize the error-prone polymerase activity in UVM-induced cells.