Abundant ribonucleotide incorporation into DNA by yeast replicative polymerases

Abstract

Measurements of nucleoside triphosphate levels in Saccharomyces cerevisiae reveal that the four rNTPs are in 36- to 190-fold molar excess over their corresponding dNTPs. During DNA synthesis in vitro using the physiological nucleoside triphosphate concentrations, yeast DNA polymerase epsilon, which is implicated in leading strand replication, incorporates one rNMP for every 1,250 dNMPs. Pol delta and Pol alpha, which conduct lagging strand replication, incorporate one rNMP for every 5,000 or 625 dNMPs, respectively. Discrimination against rNMP incorporation varies widely, in some cases by more than 100-fold, depending on the identity of the base and the template sequence context in which it is located. Given estimates of the amount of replication catalyzed by Pols alpha, delta, and epsilon, the results are consistent with the possibility that more than 10,000 rNMPs may be incorporated into the nuclear genome during each round of replication in yeast. Thus, rNMPs may be the most common noncanonical nucleotides introduced into the eukaryotic genome. Potential beneficial and negative consequences of abundant ribonucleotide incorporation into DNA are discussed, including the possibility that unrepaired rNMPs in DNA could be problematic because yeast DNA polymerase epsilon has difficulty bypassing a single rNMP present within a DNA template.

Fig. 1.

Discrimination against rNMP insertion by yeast DNA polymerases. (A) Schematic of discrimination assay. The ribonucleotide product has reduced mobility compared to the deoxynucleotide product. Reactions containing rNTPs have trace amounts of dNTPs that are incorporated (faint gray band in lane 3). (B) Results with exonuclease-deficient Pol δ. (s), substrate, (d), deoxy product, (r), ribo product. (C) Discrimination against rNMP insertion. Discrimination factors were calculated by dividing the percentage of dNTP product by the percentage of rNTP product, and then multiplying by the ratios of nucleotide concentrations and differences in enzyme concentrations and reaction times. For Pol ε, the previously characterized N-terminal catalytic fragment (49) was used. For comparison, the selectivity of RB69 (8) and ϕ29 (6), two B family polymerases are shown.

Fig. 2.

Stable incorporation of rNMPs into DNA by yeast DNA pols. (A) Alkali cleavage products of reactions with all eight NTPs at cellular concentrations are shown for Pol δ, Pol α, and Pol ε. The frequency of rNMP incorporation per nucleotide synthesized is indicated below each lane. Marker lanes on either side allow determination of the template position for rNMP incorporation. (B) Frequency of rNMP incorporation by Pol δ (green bars), Pol α (red bars), and Pol ε (blue bars) at each of 25 template positions. (C) Average frequency of rNMP incorporation by Pol δ (green bars), Pol α (red bars), and Pol ε (blue bars) according to template base identity. The largest range in rNMP incorporation frequency is shown below each template base, color-coded according to polymerase. (D) Model of a replication fork with the potential number of rNMPs incorporated by each polymerase.

Fig. 3.

Bypass of a single rNMP in a DNA template. The analysis was performed as described in Methods. The sequence on the left is that of the template strand. The X marks the location of dG or rG in the template. For the lane marked “0” min, no enzyme was added.