Acidic residues critical for the activity and biological function of yeastDNA polymerase eta

Rad30 is a member of the newly discovered UmuC/DinB/Rad30 family of DNA pol ymerases. The N-terminal regions of these proteins are highly homologous, a nd they contain five conserved motifs, I to V, while their C-terminal regio ns are quite divergent. We examined the contributions of tile C-terminal an d N-terminal regions of Rad30 to its activity and biological function. Alth ough deletion of the last 54 amino acids has no effect on DNA polymerase or thymine-thymine (T-T) dimer bypass activity, this C-terminal deletion-cont aining protein is unable to perform its biological function in vivo. The pr esence of a bipartite nuclear targeting sequence within this region suggest s that at least one function of this portion of Rad30 is nuclear targeting, To identify the active-site residues of Rad30 important for catalysis, we generated mutations of nine acidic residues that are invariant or highly co nserved among Rad30 proteins from different eukaryotic species. Mutations o f the Asp30 and Glu39 residues present in motif I and of the Asp155 residue present in motif III to alanine completely inactivated the DNA polymerase and T-T dimer bypass activities, and these mutations did not complement the UV sensitivity of the rad30 Delta mutation. Mutation of Glu156 in motif II I to alanine confers a large reduction in the efficiency of nucleotide inco rporation, whereas the remaining five Rad30 mutant proteins retain wild-typ e levels of DNA polymerase and T-T dimer bypass activities. From these obse rvations, we suggest a role for the Asp30, Glu39, and Asp155 residues in th e binding of two metal ions required for the reaction of the incoming deoxy nucleoside 5 ' -triphosphate with the 3 ' -hydroxyl in the primer terminus, while Glu156 may participate in nucleotide binding.