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

Abstract

When DNA is damaged, replicative DNA polymerases such as E. coli DNA polymerase (pol) III cannot continue replication past this damage. One of the pathways in the cell 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 UmuC, the polymerase subunit. UmuD?2C is specialized to bypass abasic sites and thymine-thymine dimers, which are the products of UV light, while DinB specializes in bypass of N2-dG adducts such as N2-furfuryl-dG. Y family polymerases can also replicate undamaged DNA in an error-prone manner and therefore play a role in mutagenesis and possibly antibiotic resistance. Using hydroxylamine, we generated a library of UmuC mutations from which we then selected those that had the ability to confer survival to nitrofurazone, which is believed to cause predominantly N2-furfuryl-dG lesions. We tested in vivo the ability of these selected UmuC variants to confer survival after UV irradiation, as well as their ability to confer the cold sensitivity phenotype and to inhibit RecA-mediated homologous recombination. In addition, further characterization suggests that some of these variants disrupt interactions with UmuD.