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A DNA-binding domain in the C-terminal region of Cdt2 enhances the DNA synthesis-coupled CRL4Cdt2 ubiquitin ligase activity for Cdt1.

The Cullin-RING ubiquitin ligase CRL4Cdt2 maintains genome integrity by mediating the cell cycle- and DNA damage-dependent degradation of proteins such as Cdt1, p21 and Set8. Human Cdt2 has two regions, a conserved N-terminal seven WD40 repeat region and a less conserved C-terminal region. Here, we showed that the N-terminal region is sufficient for complex formation with CRL4, but the C-terminal region is required for the full ubiquitin ligase activity. UV irradiation-induced polyubiquitination and degradation of Cdt1 were impaired in Cdt2 (N-terminus only)-expressing cells. Deletion and mutation analysis identified a domain in the C-terminal region that increased ubiquitination activity and displayed DNA-binding activity. The identified domain mediated binding to double-stranded DNA and showed higher affinity binding to single-stranded DNA. As the ligase activity of CRL4Cdt2 depends on proliferating cell nuclear antigen (PCNA) loading onto DNA, the present results suggest that the DNA-binding domain facilitates the CRL4Cdt2-mediated recognition and ubiquitination of substrates bound to PCNA on chromatin.