Cloning and Protein Expression Studies of an E. coli SOS Response Gene

The bacterial SOS response is a cellular reaction to environmental stress conditions such as DNA damage, high hydrostatic pressure, nutrient limitations, exposure to antibiotics etc. Induction of SOS directly up-regulates many genes responsible for various cellular processes including inhibition of cell division and non-mutagenic DNA repair. In Eschericha. coli the LexA repressor protein coordinately regulates SOS expression. The crucial point for turning on the SOS response is the interaction between the LexA and RecA recombinase. During DNA damage RecA polymerizes onto ssDNA forming nucleoprotein filament. LexA interacts with this activated RecA and is inactivated through an autoproteolysis event, resulting in induction of SOS. The SOS response directly controls the expression of approximately 45 genes. Using microarray technique after mitomycin C treatment, it has been shown that the SOS response involves gene expression changes of over 1000 gene (Khil P. P. et. al., 2002). One of the genes up-regulated after mitomycin C treatment is yjhC. Although, the biochemical function of this protein is unknown, computational annotations suggest that the yjhC is a putative dehydrogenase (Khil P. P. et. al., 2002). In the present study we are cloning the yjhC gene into an expression vector (pET21D). Once cloned, the protein expression and small scale induction tests will be performed. The expression vector will be used to purify the protein from E. coli cells, that will be used to identify the biochemical function of this protein. This work was supported by NIH grants 3R25 GM048998-13, P20 RR016480, and SC2 GM083697.