The ASCC2 CUE domain contacts adjacent ubiquitins to recognize K63-linked polyubiquitin

Alkylation of DNA and RNA is a potentially toxic lesion that can result in mutations and cell death. In response to alkylation damage, K63-linked polyubiquitin chains are assembled that localize the ALKBH3-ASCC repair complex to damage sites in the nucleus. The protein ASCC2, a subunit of the ASCC complex, selectively binds K63-linked polyubiquitin chains using its CUE domain, a type of ubiquitin-binding domain that typically binds monoubiquitin and does not discriminate among different polyubiquitin linkage types. We report here that the ASCC2 CUE domain selectively binds K63-linked diubiquitin by contacting both the distal and proximal ubiquitin. Whereas the ASCC2 CUE domain binds the distal ubiquitin in a manner similar to that reported for other CUE domains bound to a single ubiquitin, the contacts with the proximal ubiquitin are unique to ASCC2. The N-terminal portion of the ASCC2 1 helix, including residues E467 and S470, contributes to the binding interaction with the proximal ubiquitin of K63-linked diubiquitin. Mutation of residues within the N-terminal portion of the ASCC2 1 helix decreases ASCC2 recruitment in response to DNA alkylation, supporting the functional significance of these interactions during the alkylation damage response.