Anti-Rad18 Antibody (A353)

The DNA replication machinery stalls at damaged sites on templates, but normally restarts by switching to a specialized DNA polymerase(s) that carries out translesion DNA synthesis (TLS). In human cells, DNA polymerase eta (poleta) accumulates at stalling sites as nuclear foci, and is involved in ultraviolet (UV)-induced TLS. Here we show that poleta does not form nuclear foci in RAD18(-/-) cells after UV irradiation. Both Rad18 and Rad6 are required for poleta focus formation. In wild-type cells, UV irradiation induces relocalization of Rad18 in the nucleus, thereby stimulating colocalization with proliferating cell nuclear antigen (PCNA), and Rad18/Rad6-dependent PCNA monoubiquitination. Purified Rad18 and Rad6B monoubiquitinate PCNA in vitro. Rad18 associates with poleta constitutively through domains on their C-terminal regions, and this complex accumulates at the foci after UV irradiation. Furthermore, poleta interacts preferentially with monoubiquitinated PCNA, but poldelta does not. These results suggest that Rad18 is crucial for recruitment of poleta to the damaged site through protein-protein interaction and PCNA monoubiquitination.

Postreplication repair functions in gap-filling of a daughter strand on replication of damaged DNA. The yeast Saccharomyces cerevisiae Rad18 protein plays a pivotal role in the process together with the Rad6 protein. Here, we have cloned a human homologue of RAD18, hRAD18. It maps on chromosome 3p24-25, where deletions are often found in lung, breast, ovary, and testis cancers. In vivo, hRad18 protein binds to hHR6 protein through a conserved ring-finger motif. Stable transformants with hRad18 mutated in this motif become sensitive to UV, methyl methanesulfonate, and mitomycin C, and are defective in the replication of UV-damaged DNA. Thus, hRAD18 is a functional homologue of RAD18.