2013  •   Physical and LIfe Sciences

Point mutations in Escherichia coli DNA pol V that confer resistance to non-cognate DNA damage

Lead Presenter: Lisa Hawver

When DNA is damaged, replicative DNA polymerases such as E. coli DNA polymerase (pol) III stall at the site of damage and DNA replication is inhibited. One of the pathways in E. coli to cope with such damage is the SOS response that regulates the expression of at least 57 genes, including umuDC and dinB, which encode the Y family DNA polymerases UmuD?2C (pol V) and DinB (pol IV), respectively. UmuD?2C (pol V) is composed of the cleaved form of UmuD, UmuD?2, and the polymerase subunit UmuC. UmuD?2CÍs cognate lesions are abasic sites as well as the UV photoproducts cyclobutane pyrimidine dimers and 6-4 thymine-thymine photoproducts, while DinB specializes in bypassing N2-dG adducts such as N2-furfuryl-dG. Y family DNA polymerases can also replicate undamaged DNA in an error-prone manner and therefore play a role in mutagenesis and antibiotic resistance. Using hydroxylamine, we generated a library of UmuC mutations from which we then selected those that had the ability to confer resistance to nitrofurazone, which is believed to cause predominantly N2-furfuryl-dG lesions. We tested in vivo and in vitro the ability of these selected UmuC variants to confer survival after UV irradiation as well their ability to bypass N2-furfuryl-dG, and discovered that the variants may not have changed specificity of Pol V, rather, they seem to disrupt important protein-protein interactions such as the one between UmuC and UmuD/D? that are essential for Pol V function, suggesting that protein interactions are important for specific lesion bypass.