American Society of Hematology

Figure 6

$Figure 6. The E3 ligase activity of RAD18 is required for FANCD2 chromatin loading. (A) The domain structure of human RAD18 is shown. HCT116 RAD18−/− cells were transfected with plasmids expressing domain deletion, point mutation, and wild-type forms of RAD18. (B) Immunoprecipitation of endogenous FANCD2 from untreated cells expressing RAD18 mutants shows that FANCD2 binds all forms of RAD18 except the RING domain point mutant C28F. (C) Cellular fractionation of cells expressing RAD18 mutants shows that FANCD2 is loaded onto chromatin during S phase in all except RAD18-deficient cells and cells expressing the C28F mutant. (D) Colony survival assay of cells expressing RAD18 mutants shows that RAD18-deficient cells and cells expressing the C28F and ΔZNF mutants are hypersensitive to MMC. Cells expressing wild-type RAD18 and the ΔSAP mutant are resistant to MMC.$

The E3 ligase activity of RAD18 is required for FANCD2 chromatin loading. (A) The domain structure of human RAD18 is shown. HCT116 RAD18^−/− cells were transfected with plasmids expressing domain deletion, point mutation, and wild-type forms of RAD18. (B) Immunoprecipitation of endogenous FANCD2 from untreated cells expressing RAD18 mutants shows that FANCD2 binds all forms of RAD18 except the RING domain point mutant C28F. (C) Cellular fractionation of cells expressing RAD18 mutants shows that FANCD2 is loaded onto chromatin during S phase in all except RAD18-deficient cells and cells expressing the C28F mutant. (D) Colony survival assay of cells expressing RAD18 mutants shows that RAD18-deficient cells and cells expressing the C28F and ΔZNF mutants are hypersensitive to MMC. Cells expressing wild-type RAD18 and the ΔSAP mutant are resistant to MMC.

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