The effect of sequence context on spontaneous Polzeta-dependent mutagenesis in Saccharomyces cerevisiae

The Polζ translesion synthesis (TLS) DNA polymerase is responsible for over 50% of spontaneous mutagenesis and virtually all damage-induced mutagenesis in yeast. We previously demonstrated that reversion of the lys2ΔA746 −1 frameshift allele detects a novel type of +1 frameshift that is accompanied by one or more base substitutions and depends completely on the activity of Polζ. These ‘complex’ frameshifts accumulate at two discrete hotspots (HS1 and HS2) in the absence of nucleotide excision repair, and accumulate at a third location (HS3) in the additional absence of the translesion polymerase Polη. The current study investigates the sequence requirements for accumulation of Polζ-dependent complex frameshifts at these hotspots. We observed that transposing 13 bp of identity from HS1 or HS3 to a new location within LYS2 was sufficient to recapitulate these hotspots. In addition, altering the sequence immediately upstream of HS2 had no effect on the activity of the hotspot. These data support a model in which misincorporation opposite a lesion precedes and facilitates the selected slippage event. Finally, analysis of nonsense mutation revertants indicates that Polζ can simultaneously introduce multiple base substitutions in the absence of an accompanying frameshift event.