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 ε 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 ε 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 ε participates in leading-strand DNA replication.Replication of the eukaryotic nuclear genome requires DNA polymerase α to initiate synthesis at origins and to initiate synthesis of Okazaki fragments on the lagging strand, allowing DNA polymerases δ (pol δ) and ε (pol ε) to then perform the bulk of chain elongation (1,2). Pol δ is implicated in lagging-strand replication (1), but the identity of the polymerase(s) that replicates the leading strand is unknown (1,2). Null alleles of the POL2 (pol ε) and POL3 (pol δ) genes are uninformative for identifying the leading-strand polymerase, because both genes are essential for normal replication. To retain replication activity while generating a distinct mutational signature in vivo that allows assignment of pol ε to leading-and/or lagging-strand replication in yeast cells, we substituted glycine for Met 644 at the Saccharomyces cerevisiae pol ε active site. Yeast pol ε with the Met 644 Gly change retains 44% of wild-type polymerase activity (Fig. 1A) and retains full 3′ exonuclease activity (Fig. 1B). A haploid pol2-M644G yeast strain grows at a rate similar to a POL2 strain (Fig. 1C), indicating that M 644 G pol ε retains substantial replicative capacity. In both its exonuclease-proficient (Fig. 1D) and exonuclease-deficient forms (Fig. 1E), M 644 G pol ε synthesizes DNA in vitro with reduced fidelity in comparison with wild-type (i.e., Met 644 ) pol ε ( Fig. 1 and table S1) (3), i.e., it is defective in discriminating against deoxynucleotide triphosphate (dNTP) misinsertion. Even the exonuclease-proficient polymerase has an elevated base-substitution error rate (Fig. 1D), indicating that despite retaining proofreading potential (Fig. 1B), M 644 G pol ε does not efficiently proofread certain mismatches, for example, T-dTMP mismatches. This is more obvious in some sequence contexts than others. Among 16 positions in the lacZ template where T to A substitutions can be detected ( fig. S1), errors are particularly prevalent at template T

Saccharomyces cerevisiae DNA polymerase δ (Pol δ) and DNA polymerase ε (Pol ε) are replicative DNA polymerases at the replication fork. Both enzymes are stimulated by PCNA, although to different levels. To understand why and to explore the interaction with PCNA, we compared Pol δ and Pol ε in physical interactions with PCNA and nucleic acids (with or without RPA), and in functional assays measuring activity and processivity. Using surface plasmon resonance technique, we show that Pol ε has a high affinity for DNA, but a low affinity for PCNA. In contrast, Pol δ has a low affinity for DNA and a high affinity for PCNA. The true processivity of Pol δ and Pol ε was measured for the first time in the presence of RPA, PCNA and RFC on single-stranded DNA. Remarkably, in the presence of PCNA, the processivity of Pol δ and Pol ε on RPA-coated DNA is comparable. Finally, more PCNA molecules were found on the template after it was replicated by Pol ε when compared to Pol δ. We conclude that Pol ε and Pol δ exhibit comparable processivity, but are loaded on the primer-end via different mechanisms.

To better understand the functions and fidelity of DNA polymerase ε (Pol ε), we report here on the fidelity of yeast Pol ε mutants with leucine, tryptophan or phenylalanine replacing Met644. The Met644 side chain interacts with an invariant tyrosine that contacts the sugar of the incoming dNTP. M644W and M644L Pol ε synthesize DNA with high fidelity, but M644F Pol ε has reduced fidelity resulting from strongly increased misinsertion rates. When Msh6-dependent repair of replication errors is defective, the mutation rate of a pol2-M644F strain is 16-fold higher than that of a strain with wild-type Pol ε. In conjunction with earlier studies of low-fidelity mutants with replacements for the homologous amino acid in yeast Pol α (L868M/F) and Pol δ (L612M), these data indicate that the active site location occupied by Met644 in Pol ε is a key determinant of replication fidelity by all three B family replicative polymerases. Interestingly, error specificity of M644F Pol ε is distinct from that of L868M/F Pol α or L612M Pol δ, implying that each polymerase has different active site geometry, and suggesting that these polymerase alleles may generate distinctive mutational signatures for probing functions in vivo.

The emerging development of antibiotic resistant bacteria calls for novel types of antibacterial agents. In this work we examined the putative antibacterial effect of purine analogs in Listeria monocytogenes. We show that, among several tested purine analogs, only 6-N-hydroxylaminopurine (6-N-HAP) reduces the viability of the Gram-positive pathogen Listeria monocytogenes. As in Bacillus subtilis, 6-N-HAP terminates expression at guanine riboswitches in L. monocytogenes hence preventing expression of their downstream genes. However, we show that the bacteriocidal effect of the compound was unlinked to the terminated expression at the guanine riboswitches. When further examining the antimicrobial effect, we observed that 6-N-HAP acts as a potent mutagen in L. monocytogenes, by increasing the mutation rate and inducing the SOS-response. Also, addition of 6-N-HAP decreased virulence gene expression by reducing both the levels and activity of the virulence regulator PrfA.

DNA polymerase ε (Pol ε) participates in the synthesis of the leading strand during DNA replication in Saccharomyces cerevisiae. Pol ε comprises four subunits: the catalytic subunit, Pol2, and three accessory subunits, Dpb2, Dpb3 and Dpb4. DPB2 is an essential gene with unclear function. A genetic screen was performed in S. cerevisiae to isolate lethal mutations in DPB2. The dpb2-200 allele carried two mutations within the last 13 codons of the open reading frame, one of which resulted in a six amino acid truncation. This truncated Dpb2 subunit was co-expressed with Pol2, Dpb3 and Dpb4 in S. cerevisiae, but this Dpb2 variant did not co-purify with the other Pol ε subunits. This resulted in the purification of a Pol2/Dpb3/Dpb4 complex that possessed high specific activity and high processivity and holoenzyme assays with PCNA, RFC and RPA on a single-primed circular template did not reveal any defects in replication efficiency. In conclusion, the lack of Dpb2 did not appear to have a negative effect on Pol ε activity. Thus, the C-terminal motif of Dpb2 that we have identified may instead be required for Dpb2 to fulfill an essential structural role at the replication origin or at the replication fork.