Specificity and Activity of Y‐family DNA Polymerases DinB and Pol Kappa

Abstract

More than 100,000 lesions are generated on DNA per cell per day due to such agents as reactive oxygen species and ultraviolet radiation. Y‐family DNA polymerases are specialized to carry out translesion synthesis by replicating past the lesion and are conserved in all domains of life. There are two Y‐family polymerases in E. coli, DinB (Pol IV) and UmuD'2C (Pol V), as well as several in humans including Pol κ (hPol κ, a DinB ortholog). Both DinB and hPol κ are known to bypass minor groove adducts of guanine. Using primer extension assays on DNA substrates containing the minor groove adduct N2‐furfuryl‐dG as well as undamaged templates we found that loop 1 residues of DinB near the catalytic site are important for activity as well as the selection of the correct nucleotide. There is a similar trend in the loop 1 region of hPol κ. In single nucleotide incorporation assays, wild‐type and variants of both polymerases show a tendency to insert incorrect dT as well as the correct dC opposite the N2‐furfuryl‐dG adduct. This is also seen for wild‐type and variants of hPol κ on an undamaged DNA template. These observations suggest that the loop 1 region of both polymerases is important in correct nucleotide incorporation during translesion synthesis. This work is supported by the American Cancer Society.