Extended Data Fig. 5: Role of yeast elf1 in TC-NER.
From: Elongation factor ELOF1 drives transcription-coupled repair and prevents genome instability
a, Indicated mutant yeast strains were serially tenfold diluted, spotted, and exposed to indicated UV-C doses. Spot assay has been performed three times with similar results. b, Schematic showing the CPD-seq method. Isolated DNA is sonicated and adaptors are ligated. CPDs are cleaved by T4 endonuclease V and APE1 nuclease to generate 3’ ends. Following denaturing of the DNA, ends are ligated to a second adaptor that allows CPD sequencing. c, Gene plot analysis of CPD-seq data for ~4500 yeast genes, ordered by transcription frequency66. Plots depict unrepaired CPDs following 2-hour repair relative to no repair for both the transcribed strand (TS) and non-transcribed strand (NTS). Each row represents approximately 10 genes. TSS=transcription start site, TTS=transcription termination site. d, Left panel: Representative gel of bulk repair of UV-induced CPD lesions in Wt and elf1∆ mutant yeast measured by T4 endonuclease V digestion and alkaline gel electrophoresis of genomic DNA isolated from UV-irradiated yeast (100 J/m2 UV-C light) after the indicated time. Right panel: Quantification of CPD repair from n = 3 WT and n = 4 elf1∆ experiments ±SEM. *P ≤ 0.05 analyzed by unpaired two-sided t-test. e, Single nucleotide resolution analysis of CPD-seq data downstream of the TSS of ~5200 yeast genes. Plots depict fraction of unrepaired CPDs following 2-hour repair relative to no repair for both TS and NTS. Nucleosome positioning data51 is shown for reference. f, Controls for UV spotting assays shown in Fig. 4d. g, Image showing repair of CPDs in the TS of the RPB2 gene for indicated yeast strains. The image was generated by converting sequencing reads aligned to RPB2 into bands. U: unirradiated cells. Nucleotide positions relative to TSS (+1) are indicated on the left. h, Left: Relative percentage of CPDs remaining within 54 bp downstream of the TSS of the RPB2 gene. Right: Relative percentage of CPDs remaining in the downstream region (69-353 bp) of the RPB2 gene. Data are presented as mean values from all CPD sites within the indicated regions (0-54 and 69-353 bp) of the RPB2 gene ±SD from one single experiment, error bars are shown for most relevant strains. n = 8 sites (left panel), and n = 73 (right panel). i, Representation of the C. elegans elof-1 genomic organization, depicting the 180 bp emc203 deletion allele generated with CRISPR-Cas9. Shaded boxes: exons, black: coding sequences. Numerical data are provided in source data .
