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

Figure 3. SCAI promotes replication‐coupled ICL repair.

Figure 3

  1. Current model of ICLpt repair in Xenopus egg extracts (Amunugama et al, 2018).
  2. pICLpt was isolated via plasmid pull down at the indicated times after incubation in Xenopus egg extract in the presence or absence of CDC7i (100 µM) and associated proteins were analyzed by immunoblotting.
  3. pICLpt was replicated in egg extracts in the presence of [α‐32P]dATP for the indicated times, and reactions were analyzed by native agarose gel electrophoresis. The p97 inhibitor NMS‐873 (p97i; 200 μM) was supplemented to the reaction where indicated. Note that in the absence of p97 activity, CMGs are no longer unloaded from the plasmid, leading to accumulation of replication intermediate products (RI) (Fullbright et al, 2016). OC, open circular; SC, supercoiled.
  4. Samples from (C) were recovered via plasmid pull down at the indicated time points as in (B) and analyzed by immunoblotting.
  5. pICLpt was replicated in mock‐ or SCAI‐C‐depleted egg extracts, and samples collected at the indicated time points were analyzed by immunoblotting.
  6. pICLpt was replicated in mock‐ or SCAI‐depleted egg extract in the presence of [α‐32P]dATP for the indicated times, and reactions were analyzed by native agarose gel electrophoresis. ΔSCAI‐C and ΔSCAI‐N denote SCAI immunodepletion with an antibody raised against the C or N terminus of SCAI, respectively; RI, replication intermediates; OC, open circular; SC, supercoiled.
  7. Schematic of pICLpt illustrating the SapI site, which is regenerated upon replication‐coupled repair (Knipscheer et al, 2009).
  8. Quantification of SapI regeneration in mock or SCAI‐depleted extracts. Note that plasmids containing a SapI site but no ICL account for ~ 5–7% of each pICLpt preparation. Primary data are shown in Fig EV2F and G. A representative of two independent experiments is shown.
  9. Schematic of intermediates and extension products generated by AflIII digest of pICLpt.
  10. pICLpt was replicated in mock‐ or SCAI‐depleted egg extract in the presence of [α‐32P]dATP for the indicated times, and reactions were digested with AflIII and analyzed on a denaturing polyacrylamide gel. Stalling points relative to the ICL site are indicated.
  11. Schematic of intermediates and extension products generated by PsiI+XhoI double digest of pICLpt.
  12. Samples in (J) were digested with PsiI+XhoI and analyzed on a denaturing polyacrylamide gel. Stalling points relative to the ICL site are indicated.
  13. Quantification of extensions with deletions in (L) at 240 min (mean ± SEM; n = 6 independent experiments; **P < 0.01; two‐tailed paired t‐test).