Fig. 2. Restrictive temperature stress induces 3’ flap-based OFM and results in alternative duplications.

(A) Somatic mutation frequencies and types as determined by WGS, in WT and rad27Δ cells grown at 30°C or 37°C for 4 h. (B) Lengths of inserted DNA sequences in duplications in rad27Δ cells grown at 30°C or 37°C for 4 h. (C) Top, diagram of classic and alternative duplications. Bottom, frequencies of classic and alternative duplications. (D) Predicted structures leading to three types of alternative duplications. Red and green lines: DNA sequences in red and green in fig. S4A; Orange lines: yellow highlighting in fig. S4A. (E) Schematic for specific labeling of 3’ flaps in genomic DNA. Green dots: dideoxyribonucleotide; red star: ³²P-deoxyribonucleotide. (F) Levels of 3’ flaps in genomic DNA from WT, rad27Δ, or rad27Δ pol3 ITD (rad27Δ-ITD) cells grown at 30°C or 37°C for 4 h. (G) Levels of 3’ flaps in genomic DNA from rad27Δ cells grown at 37°C with or without pre-treatment with Polδ. (H and I) Reconstitution assays using ³²P-labeled 3’ flap substrate S3 (H) or ³²P-labeled secondary structure-forming 3’ flap substrate S4 or S5 (I). Substrate structures are shown above a representative image of 8% denaturing PAGE. DNA substrates (S3, S4, S5), cleavage products (Cleaved S3, S4), unligated extended 3’ flap intermediates (Extended S4, S5), and ligated extended products (Ligated P3, P4, P5) are indicated. dNTP: deoxyribonucleotides.