All three DNA polymerases that replicate the yeast nuclear genome stably incorporate rNMPs into DNA during synthesis, and rNMP incorporation probability varies over a 100-fold range depending on the polymerase, the sequence context and the identity of the rNMP [18
]. The present study shows that, in addition to introducing rNMPs into the genome, these same three polymerases can also bypass rNMPs in DNA templates, with efficiencies that vary depending on the polymerase and the identity and location of the rNMP. Polymerase dependence is apparent by comparing the relative bypass efficiencies for each of the five rNMP (columns in ). Variations range from 7.4% to 85%, with Pol α being generally most efficient and Pol δ the least efficient. Bypass efficiency also depends on the identity of the rNMP (rows in ), with a 1.5-fold variation seen with Pol α (51–75%), an approximate 3-fold variation seen with Pol ε (32–85%) and a 8-fold variation seen with Pol δ (7.4–63%).
Three types of sequence context effects are also apparent. One involves approximately 8-fold differences in relative bypass efficiency depending on the identity of the rNMP when flanked by the same neighbors (). The second is a 2-fold difference in relative bypass efficiency for the same rNMP (rG) when flanked by different neighbors (). The third context effect includes differences in dNTP insertion probability as bypass proceeds from −1 through +4 (). All three polymerases share a reduced dNTP insertion probability opposite the rNMP (, position 0). Thus, a 2′-oxygen on the sugar of the templating nucleotide of the nascent base pair reduces catalytic efficiency. Similarly, with all three polymerases and all rNMP-containing templates, dNTP insertion is also problematic when the template strand rNMP is paired with the primer-terminal base (, position +1). This reduced efficiency is not surprising, because the primer-terminal base pair of replicative DNA polymerases forms one surface of the nascent base pair binding pocket, whose geometry is critical for efficient insertion [35
]. Reduced insertion is also observed at the −1 position (several examples depicted in ), where the rNMP is in the single stranded DNA immediately adjacent to the nascent base pair binding pocket and will be the next template nucleotide to be copied. The fact that a 2′-oxygen on the sugar of this nucleotide reduces insertion is generally consistent with the fact that certain amino acids in DNA polymerases interact with this nucleotide when it is uncopied and as it is moved into position for catalysis [38
]. Finally, dNTP insertion is reduced when the template-strand rNMP is embedded in the duplex template-primer at increasing distances upstream of the active site as bypass proceeds. Effects on the insertion efficiency of Pol α and Pol ε are seen for up to four base pairs (). Theoretically, pausing at any of the rNMP or any of the four subsequent positions could slow replication folk progression. A better understanding of the effects of rNMPs in DNA on synthesis should be facilitated by crystal structures of DNA polymerases bound to primer-templates containing rNMPs at various locations as bypass proceeds, as recently accomplished for bypass of a cyclobutane pyrimidine dimer by Pol η [41
We previously reported that a pol2-M644G rnh201
Δ double mutant strain accumulates rNMPs in genomic DNA, progresses more slowly through S-phase, has elevated dNTP pools and has an elevated rate of 2–5 base pair deletions in repetitive sequences [25
]. These data indicate that unrepaired rNTPs incorporated by pol2-M644G
during replication in vivo
elicit replicative stress responses and destabilize the nuclear genome. These phenotypes correlate with the difficulty Pols α, δ and ε have in bypassing rNMPs in DNA templates. Nonetheless, the single rnh201
Δ mutant strain is not sensitive to HU, it grows relatively normal and its dNTP pools are only slightly elevated [25
]. These data indicate that unrepaired rNMPs in the nuclear genome by the wild type yeast DNA replicases are tolerated relatively well. To place this tolerance in perspective, we compared the relative bypass efficiencies for rNMPs to published values for 8-oxo-G [27
], a common lesion generated by oxidative stress and considered to be strongly mutagenic but not particularly cytotoxic. The comparisons () reveal that Pol δ and Pol ε can bypass rNMPs at least as efficiently as 8-oxo-G.
The majority of the rNMP bypass efficiency values in were determined at dNTP concentrations representative of unstressed cells. These may be minimal estimates, because rnh201
Δ strains do have slightly elevated dNTP pools [25
], and the values in for Pol δ and Pol ε demonstrate that relative bypass efficiencies are increased in the presence of “stress-induced” dNTP concentrations. The increased bypass by Pol ε observed at high dNTP concentrations is also consistent with the fact that an exonuclease-deficient derivative of Pol ε copies the rA-containing template-primer 2-fold more efficiently than the exonuclease-proficient Pol ε (). Collectively, these results suggest that the proofreading exonuclease activity of Pol ε excises dNMPs inserted during rNMP bypass, and that this excision can be prevented either by inactivating the exonuclease or by promoting extension at the expense of excision.