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. 2022 Feb 14;23(4):e53639. doi: 10.15252/embr.202153639

Figure EV3. SCAI interacts with Polζ (related to Fig 4).

Figure EV3

  1. Heat map depicting the mean of the Z‐scored Log2 label‐free quantification LFQ intensity from four biochemical replicates of pCTRL and pDPCLeads replication in Xenopus egg extracts (data originally published in Larsen et al (2019)). Where indicated, Geminin was added to block DNA replication, and ubiquitin‐vinyl sulfone (Ub‐VS) was added to deplete the pool of free ubiquitin from the extracts.
  2. pDPCLeads was replicated in mock‐, SCAI‐C‐, and SCAI‐N‐depleted egg extracts in the presence of [α‐32P]dATP. Reactions were analyzed by native agarose gel electrophoresis. Red arrowheads indicate accumulation of open circular (OC) molecules caused by a TLS defect.
  3. Nascent leading strand and extension products generated upon FspI+AatII digest of pDPC. Double digestion generates shorter damaged and longer undamaged extension products. The CMG helicase is depicted in green and the crosslinked M.HpaII protein in grey.
  4. pDPC was replicated in mock‐ or SCAI‐depleted Xenopus egg extract in the presence of [α‐32P]dATP. Samples were digested with FspI+AatII and analyzed on a denaturing polyacrylamide gel. Stalling points relative to the DPC site are indicated. U, undamaged; D, damaged extension product.
  5. U2OS cells or derivative lines stably expressing GFP or GFP‐SCAI were subjected to GFP IP followed by immunoblotting with indicated antibodies.
  6. U2OS WT or SCAI KO cells that were transfected with REV3‐GFP where indicated were subjected to IP with IgG or SCAI antibody followed by immunoblotting.
  7. GFP IPs from U2OS cells transfected with indicated GFP‐AHDC1 expression constructs were analyzed by immunoblotting.
  8. Alignment of the SCAI‐interacting regions of human and Xenopus REV3 proteins. Asterisk indicates positions with a conserved amino acid; colon indicates conservation between amino acids with strongly similar properties; period indicates conservation between amino acids with weakly similar properties.
  9. Recombinant GST or GST‐xSCAI proteins incubated or not with Biotin‐xREV3 peptide (aa 1110–1179) were subjected to streptavidin pull down and analyzed by Coomassie staining.