Ubiquitylation of DNA polymerase β via atypical K27 chains signals a DNA damage response

DNA polymerase β (Polβ) stability is tightly regulated by specific binding partners that prevent its degradation by the proteasome. Ubiquitin (Ub) chains, differing in their structural arrangements (topology), contribute to distinct cellular functions. To investigate Polβ ubiquitylation, we utilized CRISPR/Cas9-modified cell lines expressing endogenous, mClover-tagged Polβ. Our results reveal that Polβ is primarily modified with ubiquitin chains linked through lysine residues 27 [Ub(K27)] and 29 [Ub(K29)]. We further observed that Ub(K27) and Ub(K29) chains impact the utilization of ubiquitin chains linked through lysine residues 6 [Ub(K6)] and 48 [Ub(K48)] upon Polβ ubiquitylation. The ubiquitin ligase TRIP12 preferentially attaches Ub(K27) and Ub(K29) chains to Polβ. While Polβ ubiquitylation with Ub(K27) appears to have a non-degradative role, mixed ubiquitin chains and Ub(K48) chains trigger Polβ degradation. Following DNA damage, Polβ and XRCC1 translocate from the cytosol to the nucleus, while ubiquitylated Polβ relocates from the chromatin to the cytosol. This translocation of Polβ, modified with Ub(K27) chains, promotes the dissociation of the Polβ/XRCC1 complex within chromatin, and oxidative stress enhances their association in the cytosol. These findings demonstrate that atypical ubiquitin chain modifications play crucial roles in DNA repair and the DNA damage response, underscoring the unexpected importance of these chain topologies in maintaining genome integrity.