Yeast DNA polymerase epsilon participates in leading-strand DNA replication

Abstract

Multiple DNA polymerases participate in replicating the leading and lagging strands of the eukaryotic nuclear genome. Although 50 years have passed since the first DNA polymerase was discovered, the identity of the major polymerase used for leading-strand replication is uncertain. We constructed a derivative of yeast DNA polymerase epsilon that retains high replication activity but has strongly reduced replication fidelity, particularly for thymine-deoxythymidine 5'-monophosphate (T-dTMP) but not adenine-deoxyadenosine 5'-monophosphate (A-dAMP) mismatches. Yeast strains with this DNA polymerase epsilon allele have elevated rates of T to A substitution mutations. The position and rate of these substitutions depend on the orientation of the mutational reporter and its location relative to origins of DNA replication and reveal a pattern indicating that DNA polymerase epsilon participates in leading-strand DNA replication.

Fig. 1

Specific activity, growth, and fidelity analyses of M⁶⁴⁴G pol ε. (A) Relative specific activity of pol ε derivatives with activated DNA. (B) Exonuclease activity of wild-type (black boxes) and M⁶⁴⁴G pol ε (gray boxes). (C) Growth curves for wild-type (solid line) and pol2-M644G (dashed line) strains. (D) Average error rates for exonuclease-proficient wild-type (black bars) and M⁶⁴⁴G pol ε (gray bars) for base substitutions (B.S.), single-base deletions (−1), single-base insertions (+1), T to A transversions (T•dT), and A to T transversions (A•dA). Asterisks denote error rates that are ≤ the indicated value. (E) As in (D) but for exonuclease-deficient pol ε. (F) T to A transversions at −36 and +147 positions in lacZ using exonuclease-proficient M⁶⁴⁴G pol ε. Sites where T to A and A to T transversions are detectable are in bold and underlined, respectively.

Fig. 2

Variation in the rates of T to A transversions by location and gene orientation. Six haploid strains were constructed containing the pol2-M644G mutant allele. URA3 was to the right (A, B, E, and F) or left (C and D) of the indicated ARS, with the coding sequence in the Watson (A, D, and E) or Crick (B, C, and F) strand. A replication fork is shown moving away from the ARS (black box) and replicating the URA3 gene. The nascent leading strand is depicted as a single, unbroken arrow, whereas nascent Okazaki fragments on the lagging strand are depicted as broken arrows. The A–T to T–A hot spots (* for A279 and ** for A686) are represented as the inferred T-dTMP mispair generated during replication.