Y-Family Dna Polymerase Human Pol Kappa is More Tolerant of Changes to Its Active Site Loop Than Dinb

DNA damage is a constant threat from both endogenous and exogenous sources. Exposure to UV radiation and reactive oxygen species can cause DNA damage. Most damage undergoes a repair process but damage that is not repaired is bypassed in a process called translesion synthesis. Y‐family DNA polymerases carry out translesion synthesis by replicating damaged DNA as they have a larger active site to accommodate bulky DNA adducts. Y‐family DNA polymerases are conserved in all domains of life. In E. coli, there are two, DinB (Pol IV) and UmuD′2 (Pol V), one in the archaeon Sulfolobus solfataricus, Dpo4, and several in humans including pol kappa, an ortholog of DinB. Both DinB and pol kappa have been shown to bypass minor groove adducts on the N2 position of deoxyguanine while Dpo4 can bypass both minor groove and major groove adducts. Although DinB and pol kappa are homologous, we have previously shown they are blocked to different extents by major groove lesions etheno‐dA and N6‐furfuryl‐dA and that a single point mutation in an active site loop in DinB eliminates discrimination against N6‐furfuryl‐dA.In this work we created chimeric polymerases by swapping the loop region adjacent to the active site using site directed mutagenesis to assess the importance of the loop in damage specificity and activity. Using primer extension assays with DNA substrates containing the minor groove adduct N2‐furfuryl‐dG, major groove adducts etheno‐dA and N6‐furfuryl‐dA, as well as undamaged templates, we found the loop regions of DinB and pol kappa near the catalytic site are important for damage bypass specificity and accuracy of nucleotide incorporation. Specifically, hPol κ is more tolerant of substitutions in its active site loops than DinB is. By creating loop swaps, an increase in the understanding of the preferential bypass of major groove adducts by DinB and hPol κ can be accomplished.Support or Funding InformationSupport from American Cancer Society Research Scholar Grant RSG‐12‐161‐01‐DMC.