DNA polymerase kappa microsatellite synthesis: Two distinct mechanisms of slippage-mediated errors

Microsatellite tandem repeats are frequent sites of strand slippage mutagenesis in the human genome. Microsatellite mutations often occur as insertion/deletion of a repeat motif (unit-based indels), and increase in frequency with increasing repeat length after a threshold is reached. We recently demonstrated that DNA polymerase kappa (Pol kappa) produces fewer unit-based indel errors within dinucleotide microsatellites than does polymerase delta. Here, we examined human Pol kappa's error profile within microsatellite alleles of varying sequence composition and length, using an in vitro HSV-tk gap-filling assay. We observed that Pol kappa displays relatively accurate synthesis for unit-based indels, using di- and tetranucleotide repeat templates longer than the threshold length. We observed an abrupt increase in the unit-based indel frequency when the total microsatellite length exceeds 28 nucleotides, suggesting that extended Pol kappa proteinDNA interactions enhance fidelity of the enzyme when synthesizing these microsatellite alleles. In contrast, Pol kappa is error-prone within the HSV-tk coding sequence, producing frequent single-base errors in a manner that is highly biased with regard to sequence context. Single-nucleotide errors are also created by Pol kappa within di- and tetranucleotide repeats, independently of the microsatellite allele length and at a frequency per nucleotide similar to the frequency of single base errors within the coding sequence. These single-base errors represent the mutational signature of Pol kappa, and we propose them a mechanism independent of homology-stabilized slippage. Pol kappa's dual fidelity nature provides a unique research tool to explore the distinct mechanisms of slippage-mediated mutagenesis.Environ. Mol. Mutagen., 2012. (c) 2012 Wiley Periodicals, Inc.