A genetic system to analyze the products of TLS through a site-specific THF lesion.

<p>(A) Sequential steps of the THF bypass assay. A double-stranded centromeric (<i>CEN</i>) plasmid containing a THF lesion at position 605 of the <i>URA3</i> gene (white rectangle), a selectable marker (<i>LEU2</i>) and the yeast replication origin (<i>ARS4</i>) is introduced into the <i>apn1Δ apn2Δ</i> yeast strain. Transformants are selected for leucine prototrophy and then replica-plated on the medium lacking uracil to identify half-sectored colonies (shown by the arrows). The Ura^- parts of the half-sectored colonies originate from cells that underwent error-prone TLS through the THF (see text for details). (B) Possible phenotypic outcomes of the THF bypass at position 605. The lesion is indicated with an “X”. The newly synthesized strands are in blue (correct incorporation) or in red (incorrect incorporation), with nucleotides across from the lesions highlighted in bold. The amino acids at the corresponding position of the protein and the resulting phenotypes (Ura⁺ or Ura^-) are listed next to the triplet sequences. (C) Identification of TLS events by the half-sectored colony phenotype. Images of two colonies exhibiting full-growth on medium lacking leucine (SC—leu) and half-growth on medium lacking uracil (SC—leu–ura) are shown. (D) A schematic showing the “bubble”-type mismatch in the control plasmid and phenotypes associated with copying of each strand. Nucleotides that differ from the wild-type <i>URA3</i> sequence are in bold. The position 605 of the <i>URA3</i> gene is marked with an asterisk. Other symbols are as in (B).