A large intermediate domain of vertebrate REV3 protein is dispensable for ultraviolet-induced translesion replication

•In vertebrate REV3, a large intermediate domain of unresolved function is inserted.•We deleted the domain of REV3-expressing plasmid and transfected it into Rev3KO-MEF.•The transformants selected show normal UV-sensitivity and levels of UV-TLS response.•These revertants present that intermediate domain is nonessential for UV-TLS.•Using thus-truncated REV3 (210 kDa) facilitates studying of its mutagenic properties. DNA polymerase ζ (pol ζ) is involved in translesion replication (translesion synthesis, TLS) and plays an essential role in embryogenesis. In adults, pol ζ triggers mutation as a result of error-prone TLS and causes carcinogenesis. The catalytic subunit of pol ζ, REV3, is evolutionarily conserved from yeast and plants to higher eukaryotes. However, the structures are notably different: unlike that in yeast REV3, a large intermediate domain is inserted in REV3 of humans and mice. The domain is mostly occupied with noncommittal structures (random coil…etc.); therefore, its role and function are yet to be resolved. Previously, we reported deficient levels of ultraviolet (UV)-induced TLS in fibroblasts derived from the Rev3-knockout mouse embryo (Rev3KO-MEF). Here, we constructed a mouse Rev3-expressing plasmid with a deleted intermediate domain (532-1793 a.a,) and transfected it into Rev3KO-MEF. The isolated stable transformants showed comparable levels of UV-sensitivity and UV-TLS activity to those in wild-type MEF, detected using an alkaline sucrose density gradient sedimentation. These results indicate that the intermediate domain is nonessential for UV-induced translesion replication in cultured mouse cells.