Evidence from mutational specificity studies that yeast DNA polymerases delta and epsilon replicate different DNA strands at an intracellular replication fork

Although polymerases delta and epsilon are required for DNA replication in eukaryotic cells, whether each polymerase functions on a separate template strand remains an open question. To begin examining the relative intracellu lar roles of the two polymerases, we used a plasmid-borne yeast tRNA gene a nd yeast strains that are mutators due to the elimination of proofreading b y DNA polymerases delta or epsilon. Inversion of the tRNA gene to change th e sequence of the leading and lagging strand templates altered the specific ities of both mutator polymerases, but in opposite directions. That is, the specificity of the polymerase delta mutator with the tRNA gene in one orie ntation bore similarities to the specificity of the polymerase epsilon muta tor with the tRNA gene in the other orientation, and vice versa. We also ob tained results consistent with gene orientation having a minor influence on mismatch correction of replication errors occurring in a wild-type strain. However, the data suggest that neither this effect nor differential replic ation fidelity was responsible for the mutational specificity changes obser ved in the proofreading-deficient mutants upon gene inversion. Collectively , the data argue that polymerases delta and epsilon each encounter a differ ent template sequence upon inversion of the tRNA gene, and so replicate opp osite strands at the plasmid DNA replication fork. (C) 2000 Academic Press.