Figure 2.
Polκ acts in FA pathway-mediated TLS during aldehyde-ICL repair. (A) Scheme of plasmid pulldown assay coupled to mass spectrometry (pp-ms). Biotinylated LacI-coupled streptavidin beads are used to pull down reaction intermediates during ICL repair. DNA-bound proteins are identified by MS analysis. (B) Relative abundance of indicated proteins is represented by a heatmap showing Z-scores (mean from four biological replicates). (C) Western blot analysis of mock, Polκ, Rev1, or Polκ/Rev1 depleted egg extract alongside a titration of undepleted extract. (D) Scheme for the formation of products detected on a sequencing gel, nascent products in grey. (E) Repair intermediates of pICL-AARED replicated in mock, Polκ, Rev1, or Polκ/Rev1 depleted egg extract supplemented with 32P-dCTP are extracted, digested with AflIII, and separated on a 7% urea–PAGE gel. Grey arrows: −1 stalling products; open arrows: 0 stalling products; white arrows: −1/0 stalling products.