| Ubiquitin (Ub) is a small, highly conserved protein that is covalently attached to lysine residues on substrate proteins, signaling in myriad eukaryotic pathways. Ub signaling is diversified in part by the capacity for lysine residues on Ub to become sites of ubiquitination, resulting in polyubiquitin (polyUb) chains. PolyUb chains linked through different lysine residues act as distinct signals, targeting the ubiquitinated substrate to different pathways and functions. For example, polyUb chains linked through lysine 48 target a modified substrate for proteasomal degradation, while polyUb chains linked through lysine 63 (K63 chains) signal nonproteolytically in a variety of pathways, including error-free DNA damage tolerance.; The enzymatic cascade that results in K63 chain assembly in the error-free damage tolerance pathway has been elucidated; however, the mechanism through which the K63 chain signal leads to DNA lesion bypass is uncharacterized.; We used alanine scanning mutagenesis to map epitopes of Ub that participate in K63 chain signaling in damage tolerance. This approach identified 15 surface residues of Ub that significantly contribute to K63 chain signaling in this pathway. The 15 residues largely cluster in several patches that likely mediate interactions with factors involved in K63 chain signal assembly and recognition in error-free DNA damage tolerance.; We also conducted a yeast two-hybrid screen searching for K63 chain interactoring proteins using an innovative linear polyUb construct as a K63 chain mimic. Although no interactors were identified in the screen, we demonstrated that the linear polyUb is recognized as a polyUb molecule by several Ub-binding proteins. Features of the linear polyUb construct, including its effective mimicry of a K63 chain and its resistance to deubiquitinases, make this a useful tool for future investigations into K63 chain binding and recognition. |
