Directed Evolution of DinB in Escherichia coli by Hydroxylamine Mutagenesis and UV Selection

Abstract

DNA polymerase III (pol III) replicates undamaged DNA in E. coli under normal conditions. However, pol III is unable to replicate past DNA damage and when this happens, the SOS response is induced. The SOS response induces the expression of at least 57 genes that help repair or tolerate the damage. Some of the genes expressed during the SOS response include those encoding the Y‐family polymerases, which are capable of translesion synthesis (TLS). Translesion synthesis is the process by which DNA polymerases replicate damaged DNA without correcting the damage.The dinB gene product (pol IV) is capable of replicating DNA containing minor groove deoxyguanine adducts, such as N2‐furfuryl‐dG and N2‐benzo[a]pyryl‐dG adducts, whereas pol V, a product of the umuC and umuD gene products, is capable of replicating DNA containing lesions caused by ultraviolet (UV) light. Our goal was to induce TLS across UV lesions with variants of pol IV. We used DinB and chimeras of DinB containing fragments of UmuC, with additional random mutations via treatment with hydroxylamine, to identify variants of DinB that have obtained the ability to replicate past UV lesions. This work is supported by the American Cancer Society.