The wild‐type Schizosaccharomyces pombe mat1 imprint consists of two ribonucleotides

Ribonucleotides are frequently incorporated into DNA during replication, they are normally removed, and failure to remove them results in replication stress. This stress correlates with DNA polymerase (Pol) stalling during bypass of ribonucleotides in DNA templates. Here we demonstrate that stalling by yeast replicative Pols δ and e increases as the number of consecutive template ribonucleotides increases from one to four. The homologous bacteriophage RB69 Pol also stalls during ribonucleotide bypass, with a pattern most similar to that of Pol e. Crystal structures of an exonuclease-deficient variant of RB69 Pol corresponding to multiple steps in single ribonucleotide bypass reveal that increased stalling is associated with displacement of Tyr391 and an unpreferred C2´-endo conformation for the ribose. Even less efficient bypass of two consecutive ribonucleotides in DNA correlates with similar movements of Tyr391 and displacement of one of the ribonucleotides along with the primer-strand DNA backbone. These structurefunction studies have implications for cellular signaling by ribonucleotides, and they may be relevant to replication stress in cells defective in ribonucleotide excision repair, including humans suffering from autoimmune disease associated with RNase H2 defects.translesion synthesis | replication stalling | DNA replication

Section: Discussionsupporting

confidence: 59%

mentioning

confidence: 99%

Structure–function analysis of ribonucleotide bypass by B family DNA replicases

Clausen

Murray

Passer

et al. 2013

“…For example, eukaryotic Pol mu is highly proficient at rNTP incorporation and is suggested to use rNTPs during nonhomologous end joining of ds breaks at low dNTP concentration (37). Indeed, it appears that ligases involved in nonhomologous end joining are more proficient in using a 3′ terminal rNMP (38,39). Moreover, in S. pombe, two rNMPs are incorporated in S phase and retained during the next round of replication, which enhances the mating type switch recombination event (39).…”

Section: Discussionmentioning

confidence: 99%

“…Indeed, it appears that ligases involved in nonhomologous end joining are more proficient in using a 3′ terminal rNMP (38,39). Moreover, in S. pombe, two rNMPs are incorporated in S phase and retained during the next round of replication, which enhances the mating type switch recombination event (39). It has been hypothesized by the Kunkel laboratory that the high frequency of rNMP incorporation by yeast replicases may be harnessed for DNA metabolic actions, including use as marks of the newly replicated strand of DNA (2).…”

Section: Discussionmentioning

confidence: 99%

Cost of rNTP/dNTP pool imbalance at the replication fork

Yao

Schroeder

Yurieva

et al. 2013

106

188

The concentration of ribonucleoside triphosphates (rNTPs) in cells is far greater than the concentration of deoxyribonucleoside triphosphates (dNTPs), and this pool imbalance presents a challenge for DNA polymerases (Pols) to select their proper substrate. This report examines the effect of nucleotide pool imbalance on the rate and fidelity of the Escherichia coli replisome. We find that rNTPs decrease replication fork rate by competing with dNTPs at the active site of the C-family Pol III replicase at a step that does not require correct base-pairing. The effect of rNTPs on Pol rate generalizes to B-family eukaryotic replicases, Pols δ and e. Imbalance of the dNTP pool also slows the replisome and thus is not specific to rNTPs. We observe a measurable frequency of rNMP incorporation that predicts one rNTP incorporated every 2.3 kb during chromosome replication. Given the frequency of rNMP incorporation, the repair of rNMPs is likely rapid. RNase HII nicks DNA at single rNMP residues to initiate replacement with dNMP. Considering that rNMPs will mark the new strand, RNase HII may direct strand-specificity for mismatch repair (MMR). How the newly synthesized strand is recognized for MMR is uncertain in eukaryotes and most bacteria, which lack a methyl-directed nicking system. Here we demonstrate that Bacillus subtilis incorporates rNMPs in vivo, that RNase HII plays a role in their removal, and the RNase HII gene deletion enhances mutagenesis, suggesting a possible role of incorporated rNMPs in MMR.T he structures of ribonucleoside triphosphates (rNTPs) and deoxyribonucleoside triphosphates (dNTPs) differ by a single atom, yet each must be distinguished by DNA and RNA polymerases (Pols). This is more challenging for DNA Pols because the intracellular concentration of rNTPs is 10-100-fold higher than that of dNTPs (1-3). DNA is more stabile than RNA, and rNMP residues in DNA could lead to spontaneous strand breaks. Hence, it is important to genomic integrity that DNA Pols exclude rNMPs from incorporation into the genome (1, 4).Structural studies reveal that DNA Pols distinguish ribo and deoxyribo sugars via a "steric gate," in which a bulky residue or main chain atom sterically occludes binding of the ribo 2′OH (5, 6). However, the single-atom difference between rNTPs and dNTPs imposes an upper limit to sugar recognition, and thus DNA Pols incorporate rNMPs at a low frequency. For example, studies in yeast demonstrate that rNMPs are incorporated in vivo, and studies in vitro demonstrate a frequency of rNMP incorporation predicting that 10,000 rNMPs or more may be incorporated each replication cycle (2,7,8). Given their abundant incorporation, there are probably multiple pathways to remove rNMPs, as their persistence is associated with genomic instability in yeast (7, 9).The current study reconstitutes the Escherichia coli replisome and examines the cost of rNTP/dNTP nucleotide pool imbalance on the frequency of rNMP incorporation and the rate of fork progression. We find that a nucleotide pool imbalance slows the ...

“…The imprint is maintained until the following S phase, where it stalls leading strand replication, thereby inducing recombination that leads to mating-type switching (see ref. 39 and references therein). One possible origin for a ribonucleotide imprint is lagging strand DNA replication, perhaps involving RNA primase or rNMP incorporation by Pol α, which has the highest "per-nucleotide" rNMP incorporation capacity of the three replicative polymerases (Fig.…”

mentioning

confidence: 99%

Abundant ribonucleotide incorporation into DNA by yeast replicative polymerases

McElhinny

Watts

Kumar

et al. 2010

377

582

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 ε, which is implicated in leading strand replication, incorporates one rNMP for every 1,250 dNMPs. Pol δ and Pol α, 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 α, δ, and ε, 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 ε has difficulty bypassing a single rNMP present within a DNA template.DNA replication | nucleotide precursors | nucleotide selectivity T he integrity of the eukaryotic genome is ensured in part by the chemical nature of the storage medium-DNA. Compared to RNA, DNA is inherently more resistant to strand cleavage due to the absence of a reactive 2′ hydroxyl on the ribose ring. The active sites of most DNA polymerases are evolved to efficiently exclude ribonucleoside triphosphates (rNTPs) from being incorporated during DNA synthesis (reviewed in (1)). However, rNTP exclusion is not absolute. Early studies (reviewed in (1, 2)) revealed that DNA polymerases do incorporate rNMPs during DNA synthesis. Kinetic studies (3-13) have further demonstrated that selectivity for insertion of dNMPs into DNA rather than rNMPs varies from 10-fold to >10 6 -fold, depending on the DNA polymerase and the dNTP/rNTP pair examined. rNMP incorporation during DNA synthesis is potentially made more probable by the fact that the concentrations of rNTPs in vivo are higher than are the concentrations of dNTPs (e.g., see refs. 2, 14 and results of this study). Thus some rNMPs are likely to be stably incorporated into DNA during replication, and possibly during DNA repair, e.g., nonhomologous end joining (NHEJ) of double strand breaks in DNA (9, 15). This possibility is supported by biochemical studies implicating RNase H2 and FEN1 in the repair of single ribonucleotides in DNA (16,17). It is therefore of interest to know just how frequently rNMPs are incorporated into DNA by the DNA polymerases that synthesize the most DNA in a eukaryotic cell, namely DNA polymerases α, δ, and ε. Here we investigate this by first measuring the rNTP and dNTP concentrations in budding yeast. We then use these concentrations in DNA sy...