Proofreading of ribonucleotides inserted into DNA by yeast DNA polymerase ɛ

Abstract

We have investigated the ability of the 3' exonuclease activity of Saccharomyces cerevisiae DNA polymerase ɛ (Pol ɛ) to proofread newly inserted ribonucleotides (rNMPs). During DNA synthesis in vitro, Pol ɛ proofreads ribonucleotides with apparent efficiencies that vary from none at some locations to more than 90% at others, with rA and rU being more efficiently proofread than rC and rG. Previous studies show that failure to repair ribonucleotides in the genome of rnh201Δ strains that lack RNase H2 activity elevates the rate of short deletions in tandem repeat sequences. Here we show that this rate is increased by 2-4-fold in pol2-4 rnh201Δ strains that are also defective in Pol ɛ proofreading. In comparison, defective proofreading in these same strains increases the rate of base substitutions by more than 100-fold. Collectively, the results indicate that although proofreading of an 'incorrect' sugar is less efficient than is proofreading of an incorrect base, Pol ɛ does proofread newly inserted rNMPs to enhance genome stability.

Fig. 1. Ribonucleotide extension, incorporation and proofreading by Pol ε

(A) Sequences of primer-templates used for panel B (top two substrates) and panel C (lower substrate); (B) Alkali-cleavage of extension products. ε(+) and ε(−) refer to proofreading-proficient and proofreading-deficient Pol ε, respectively. NE indicates the no enzyme control. For the lanes under dC, the highest mobility band represents the unextended deoxy-terminated primer (d-OH). For lanes under rC, the highest mobility band (r-PO₄) represents the 3′-terminal phosphate-containing product of extension followed by alkali cleavage. This molecule migrates faster due to the presence of the terminal-phosphate [19]. The percentage of alkali-resistant product is indicated below the image; (C) Stable rNMP incorporation. Lanes marked (U) depict products generated by Pol ε prior to gel purification, as described in [5]. The percentage of alkali sensitive products and the percentage of rNMP incorporation per nucleotide synthesized are shown below each lane. The mean and standard deviation for triplicate measurements was 2.1 ± 0.3 for wild type Pol ε and 3.1 ± 0.02 for exonuclease-deficient Pol ε; (D) Average frequency of ribonucleotide incorporation for rU, rA, rC and rG calculated from (C). The relative difference in ribonucleotide incorporation between proofreading-proficient and –deficient Pol ε is shown above each base; (E) Proofreading efficiency calculated as 1-(rNMP incorporation for proofreading proficient pol ε/rNMP incorporation for proofreading deficient pol ε) at 24 template positions; (F) Proofreading at two C and two T in four different sequence contexts: C57, C51, T60 and T52. The template base located at the site of proofreading is between the two spaces. G and C bases are underlined and in bold face to highlight the possibility that increased G+C content suppresses proofreading.

Fig. 2. Phenotypic analysis of the pol2–4 rnh201Δ strain

Ten-fold serial dilutions of exponentially growing cells from the indicated strains were grown on YPDA (untreated) or exposed to 150 mM hydroxyurea (HU). Plates were incubated at 30°C for 3 days and photographed. A pol2-M644G rnh201Δ mutant strain that shows moderate sensitivity to HU was included as a positive control.

Fig. 3. URA3 mutational spectrum for the pol2–4 rnh201Δ strain

The coding strand of the 804 base pair URA3 open reading frame is shown with every tenth base indicated by a circle below the DNA sequence. The sequence changes observed upon sequence analysis of independent ura3 mutants are depicted above the coding sequence for URA3 orientation 1 in red, and below the coding sequence for URA3 orientation 2 in blue. Letters indicate single base substitutions, closed triangles indicate single base additions, open triangles indicate single base deletions, and short lines indicate deletions of 2–5 base pairs.

Fig. 4. Specific mutation rates and proofreading factor for URA3 mutation classes

The mutation rate and proofreading values corresponding to these graphs are listed in Supplemental Tables S3 and S4; (A) Mutation rates and proofreading factors for the POL2⁺ rnh201Δ and pol2–4 rnh201Δ strains. Mutation rates for the indicated deletion mutations were calculated using the data in Table 1 and Fig. 3 as the fraction of each type of event among the total mutants sequenced, multiplied by the overall mutation rate for each strain. Rates for the POL2⁺ rnh201Δ strain were calculated from the URA3 spontaneous mutation rates in Table 1 and previously collected mutational spectra [11]. Proofreading factors were calculated by dividing the rate for the indicated mutation in the pol2–4 rnh201Δ strain by the rate for that mutation in the POL2⁺rnh201Δ mutant (Table 1 and Fig. 3; [11]). The orientation of the URA3 reporter (OR1 or OR2) is indicated. The asterisk (*) indicates that no events were observed for the pol2–4 rnh201Δ strain; (B) Mutation rates and proofreading factors for the indicated base substitution hotspots were calculated for the POL2⁺ rnh201Δ and pol2–4 rnh201Δ strains as described in part (A). Data correspond to strains in which URA3 is in orientation 1 (OR1).