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

• Published in: Journal of molecular biology 2000-06, Vol.299 (2), p.405-419
• Main Authors: Karthikeyan, R, Vonarx, E.J, Straffon, A.F.L, Simon, M, Faye, G, Kunz, B.A
• Format: Article
• Language: English
• Subjects: Alleles; Base Pair Mismatch - genetics; Base Sequence; Centromere - genetics; centromeres; Chromosome Inversion; deletions; dna mismatch repair; DNA Polymerase II - genetics; DNA Polymerase II - metabolism; DNA Polymerase III - genetics; DNA Polymerase III - metabolism; DNA Repair - genetics; DNA replication; DNA Replication - genetics; DNA, Fungal - biosynthesis; DNA, Fungal - genetics; DNA, Single-Stranded - genetics; DNA-directed DNA polymerase; Exodeoxyribonucleases - genetics; Exodeoxyribonucleases - metabolism; genes; Genes, Fungal - genetics; insertions; Kinetics; Molecular Sequence Data; Mutagenesis - genetics; mutants; mutation; Nucleic Acid Heteroduplexes - genetics; plasmids; Plasmids - genetics; RNA, Transfer - genetics; Saccharomyces cerevisiae; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae - genetics; Substrate Specificity; Templates, Genetic; transfer RNA
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1006/jmbi.2000.3744

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 intracellular roles of the two polymerases, we used a plasmid-borne yeast tRNA gene and yeast strains that are mutators due to the elimination of proofreading by DNA polymerases delta or epsilon. Inversion of the tRNA gene to change the sequence of the leading and lagging strand templates altered the specificities of both mutator polymerases, but in opposite directions. That is, the specificity of the polymerase delta mutator with the tRNA gene in one orientation bore similarities to the specificity of the polymerase epsilon mutator with the tRNA gene in the other orientation, and vice versa. We also obtained 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 replication fidelity was responsible for the mutational specificity changes observed in the proofreading-deficient mutants upon gene inversion. Collectively, the data argue that polymerases delta and epsilon each encounter a different template sequence upon inversion of the tRNA gene, and so replicate opposite strands at the plasmid DNA replication fork.