Figure 3.
Polκ promotes insertion for aldehyde ICLs and extension for cisplatin ICLs. (A) Western blot analysis of mock, Polκ-depleted (ΔPolκ), and Polκ-depleted egg extract supplemented with recombinant WT Polκ (ΔPolκ + WT), alongside a titration of undepleted extract. (B) Repair intermediates of pICL-AARED, pICL-ACRRED, and pICL-Pt replicated in mock, ΔPolκ, and ΔPolκ + WT egg extract supplemented with 32P-dCTP were 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. The stalled products of the rightward fork at Pt-ICLs are 2 nt longer compared to the aldehyde ICLs due to a difference in ICL position as described in Fig. 1F. (C) Schematic representation of the lesion bypass assay. Replication/repair intermediates are digested with HincII and separated on a denaturing agarose gel. Long and short arms appear as the replication forks approach the lesion and are converted to full-length products following TLS and HR. An undamaged plasmid (pQuant) is added to the reactions for quantification. Quantification of lesion bypass assays on pICL-AARED (D), pICL-ACRRED (E), and pICL-Pt (F) repair reactions in mock, ΔPolκ, and ΔPolκ + WT egg extract. Based on gels in Supplementary Fig. S5G–I. (G) Repair intermediates of pICL-ACRRED replication reactions in mock, ΔPolκ, and ΔPolκ + WT egg extracts were digested with HincII, or HincII and PmlI, and separated on agarose gel (Supplementary Fig. S5J). Repair was calculated based on the regeneration of the PmlI recognition site (left scheme) and plotted (right). (H) Similar to (G) but for pICL-Pt, containing a SapI restriction site (left scheme) that is regenerated upon repair. Based on gel in Supplementary Fig. S5K.