Using fragments of human c-Ha-ras and mouse Ha-ras1 genes containing 8-hydroxyguanine (8-OH-G) in hypermutagenic codon 12, we analyzed the kinetics of DNA synthesis catalyzed by human Polkappa. This translesion DNA polymerase, belonging to the Y-family, was found to be moderately inhibited by the presence of 8-OH-G on either mouse or human templates. From our previous results, inhibition of various polymerases by 8-OH-G increases in the following order: Poleta < Polkappa < Polbeta < Polalpha, showing that major replicative and repair polymerases are more sensitive to this lesion than enzymes belonging to the Y-family. In the direct mutagenesis experiments, Polkappa was found to be more mutagenic than Poleta studied previously: it inserted dAMP more efficiently than dCMP opposite 8-OH-G. Polkappa was also able to cause indirect mispair ('action-at-a-distance' mutagenesis), this effect being more distinct on mouse templates. Two adjacent 8-OH-G residues in codon 12 inhibited Polkappa moderately and induced misincorporation of dAMP. However, this effect was not comparable to the strong relaxation of the enzyme specificity, observed previously in the case of Poleta. Polkappa catalyzed incorporation (and misincorporation of dAMP) much more efficiently on mouse templates, human DNA fragments being distinctly worse substrates. Interestingly, in direct mutagenesis systems, the preference for dAMP over dCMP was nearly the same on mouse and human templates.
Mammalian DNA polymerase kappa (pol kappa), a member of the UmuC/DinB nucleotidyl transferase superfamily, has been implicated in spontaneous mutagenesis. Here we show that human pol kappa copies undamaged DNA with average single-base substitution and deletion error rates of 7 x 10(-3) and 2 x 10(-3), respectively. These error rates are high when compared to those of most other DNA polymerases. pol kappa also has unusual error specificity, producing a high proportion of T.CMP mispairs and deleting and adding non-reiterated nucleotides at extraordinary rates. Unlike other members of the UmuC/DinB family, pol kappa can processively synthesize chains of 25 or more nucleotides. This moderate processivity may reflect a contribution of C-terminal residues, which include two zinc clusters. The very low fidelity and moderate processivity of pol kappa is novel in comparison to any previously studied DNA polymerase, and is consistent with a role in spontaneous mutagenesis.
