Replication of ribonucleotide-containing DNA templates by yeast replicative polymerases

Abstract

The major replicative DNA polymerases of S. cerevisiae (Pols α, δ, and ɛ) incorporate substantial numbers of ribonucleotides into DNA during DNA synthesis. When these ribonucleotides are not removed in vivo, they reside in the template strand used for the next round of replication and could potentially reduce replication efficiency and fidelity. To examine if the presence of ribonucleotides in a DNA template impede DNA synthesis, we determined the efficiency with which Pols α, δ, and ɛ copy DNA templates containing a single ribonucleotide. All three polymerases can replicate past ribonucleotides. Relative to all-DNA templates, bypass of ribo-containing templates is slightly reduced, to extents that depend on the identity of the ribo and the sequence context in which it resides. Bypass efficiencies for Pols δ and ɛ were increased by increasing the dNTP concentrations to those induced by cellular stress, and in the case of Pol ɛ, by inactivating the 3'-exonuclease activity. Overall, ribonucleotide bypass efficiencies are comparable to, and usually exceed, those for the common oxidative stress-induced lesion 8-oxo-guanine.

Figure 1. PAGE phosphorimages of the bypass of a single rNMP by Pols α, δ and ε

Gel image of DNA products for primer extension reactions with (A) Pol α using substrates 1–6 (B) Pol δ using substrates 1–6 and (C) Pol ε using substrates 7–12. The template sequence is shown to the left of the image and the arrow depicts the location of full-length product. The “R” represents the location of the corresponding rNMP in the template. No enzyme was added to the un-extended primer (designated 0 min). The products of reactions incubated for 20 minutes were not used for quantification, and are shown only to illustrate that some bypass occurs with all six templates. (C) The boxed lane for the 5′-rG template at 20 min depicts an increase in the grey scale to show the presence of full-length product. For the rA- and rU-containing templates, some products of the 6-minute reactions resulted from multiple cycles of extension, so this time point was not used for quantification. Additional control reactions were performed to compare the efficiency of bypass of four different all-DNA control templates, containing dG, dC, dA or dT at the position corresponding to the rNMP. Variations in bypass efficiencies among these controls were 2-fold or (usually) less (data not shown). Thus, results for the control template shown here were used to calculate relative bypass efficiencies for all five rNMP-containing templates (Table 2).

Figure 2. Polymerase termination probability during rNMP bypass

Gel images of reaction products shown in Fig. 1 were quantified as described in Methods. Bar graph of termination probability (vertical axis, 0–100%) at each incorporation (horizontal axis) for (A) Pol α, (B) Pol δ, and (C) Pol ε. Position “0” corresponds to the location of the rNMP in (A), −1 indicates the preceding incorporation, and +1 through +4 indicate sequential incorporations after insertion at “0”. Error bars represent the standard deviations. The arrows indicate the values for reactions with 10-fold cellular dNTP concentration.