Rad18 binds SHPRH and HLTF through its ubiquitin-binding ZnF domain. (A) Domain structure of Rad18 showing wild-type, Rad18 ΔZnF(Δ200–224), and Rad18-ZnF(186–240) constructs (Huang et al., 2009). BD, binding domain. (B) The Rad18-ZnF is important to bind SHPRH. FLAG-Rad18 constructs lacking the indicated domains were cotransfected with GFP-SHPRH. FLAG-Rad18 and interacting proteins were analyzed as in Fig. 1 B. (C) The Rad18-ZnF contributes to SHPRH binding. GST–Rad18-ZnF or full-length Rad18 was used to pull-down GFP-SHPRH from lysates and analyzed as in Fig. 1 C. (D) The Rad18-ZnF interacts directly with the SHPRH-H15 domain. GST–SHPRH-H15 was used to pull-down FLAG–Rad18-ZnF from cell lysates and analyzed as in Fig. 1 C. (E) The Rad18-ZnF is important for binding HLTF. FLAG-Rad18 constructs lacking the indicated domains were cotransfected with untagged HLTF. FLAG-Rad18 and interacting proteins were analyzed as in Fig. 1 B. (F) HLTF binding is disrupted by the Rad18-Ub fusion. GST-Rad18 fusion proteins were incubated with cell lysates transfected with GFP-HLTF and analyzed as in Fig. 1 C.