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Chf protein autoubiquitination is RING-dependent and FHA-independent. Chf RING mutants “R” (Chf1-C345S, H350A and Chf2-C451S, H456A) and FHA mutants “F1” (Chf1-G192E and Chf2-G298E) and “F2” (Chf1-S220A, H223L and Chf2- S326A, H329L) were tested for autoubiquitination activity with Ubc4 (A) or Ubc13/Mms2 (B) supplied as the E2. In these assays, RING domain mutants are undistinguishable from omitting the E3 ligase, whereas FHA domain mutants are undistinguishable from wild-type.
