Figure 2. Effect of mutations in XPF‐ERCC1 on ICL repair in Xenopus egg extract.

• A
  Schematic representation of repair of a plasmid containing a site‐specific cisplatin ICL (pICL) in Xenopus egg extract. The SapI site that is blocked by the ICL becomes available on one of the replicated molecules after full repair via HR using the sister molecule (Fig EV1). The sister molecule is repaired by lesion bypass, but retains the unhooked ICL that is not removed efficiently in Xenopus egg extract (Räschle et al, 2008).
• B
  XPF‐ERCC1‐depleted (ΔXE) and XPF‐ERCC1‐depleted extracts complemented with wild‐type (XE^WT) or indicated mutant XPF‐ERCC1 (XE^MUT) were analyzed by Western blot using α‐XPF antibodies (left panel). Line within blot indicates position where irrelevant lanes were removed. These extracts were used to replicate pICL. Replication intermediates were isolated and digested with HincII, or HincII and SapI, and separated on agarose gel. Repair efficiency, represented by SapI regeneration, was calculated as described (Räschle et al, 2008) and plotted (right panel).
• C, D
  As in (B) but analyzing different XPF‐ERCC1 mutant complexes. Experimental replicates are shown in Fig EV3. Note: repair levels can differ per batch of individually prepared extract or per depletion experiment and can only be compared within an experiment. *, background band.

Data information: (B–D) #, SapI fragments from contaminating uncrosslinked plasmid present in varying degrees in pICL preparations.Source data are available online for this figure.