Analyses of ultraviolet-induced focus formation of hREV1 protein

Translesional DNA synthesis (TLS) is one of the DNA damage tolerance mechanisms that allow cells with DNA damage to continue DNA replication. Each of the mammalian Y-family DNA polymerases (Pol eta, Pol iota, Pol kappa, and REV1) has been shown to carry out TLS by itself or in combination with another enzyme in vitro. Recently, the C-terminal region of mammalian REV1 (the total 1251 residues in human) was found to interact with Pol eta, Pol iota, and Pol kappa, as well as with the REV7 subunit of another TLS enzyme, Pol zeta. Thus, it is proposed that REV1 plays a pivotal role in TLS in vivo. We here describe our study on the localization of human REV1 protein (hREV1) in nondamaged and ultraviolet (UV)-irradiated cells. Ectopically expressed hREV1 in mammalian cells was localized to the nucleus and exhibited dozens of tiny foci in approximately 3% of nondamaged cells. The percentage of focus-forming cells markedly increased after UV irradiation in a time- and dose-dependent manner. The focus formation was associated with UV-induced DNA damage. Interestingly, although the hREV1 foci in S-phase cells colocalized with PCNA foci, suggesting the association of hREV1 with the replication machinery, hREV1 focus formation was observed not only in the S phase but also outside S phase. Furthermore, it was found that the hREV1 focus formation after UV irradiation required a region near the C-terminal (826-1178).