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

Abstract

The Polzeta 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 lys2DeltaA746 -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 Polzeta. 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 Poleta. The current study investigates the sequence requirements for accumulation of Polzeta-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 Polzeta can simultaneously introduce multiple base substitutions in the absence of an accompanying frameshift event.

Figure 1.

Reversion spectra of the lys2ΔA746 allele in presence (RAD30) and absence (rad30Δ) of Polη. Spectra were generated in an NER-defective (rad14Δ) background and the number of revertants sequenced (N) is indicated next to the strain genotype. The nt deleted to create the lys2Δ▵A746 allele is indicated by ‘—’, and base substitutions introduced to extend the reversion window are indicated by lowercase letters (30). Insertions of a single nt are shown as ‘+’ and complex insertions (‘cins’) are indicated above the sequence; complex deletions (‘cdel’) are shown below the sequence. Deletions of 95 bp or 131 bp with endpoints in short direct repeats are indicated as ‘large DEL’ and are above each spectrum. The boxed regions represent HS1, HS2 and HS3.

Figure 2.

Sequence changes to the lys2ΔA746 reversion window. The positions of the 6A run (boxed), the unique BglII site where the transposed hotspots sequences were inserted (underlined) and the core HS1-HS3 sequences (boxed) are indicated. The sequences of the insertions or base substitutions used to create specific alleles are indicated below the reversion window sequence. Lowercase letters indicate the site-specific sequence changes.

Figure 3.

Summary of sequence alterations to the lys2ΔA746 reversion window. The relative positions of key elements (HS1–HS3, transposed hotspots, the mutated 6A run, the ΔA746 mutation) within the 150-bp reversion window are indicated. An ‘X’ through an element indicates its elimination by site-directed mutagenesis. The allele names that correspond to the sequence changes are indicated to the left of each cartoon and the relevant results are summarized to the right.