Maintaining the chemical identity of DNA depends on ribonucleotide exclusion by DNA polymerases. However, ribonucleotide exclusion during DNA synthesis in vitro is imperfect. To determine if ribonucleotides are incorporated during DNA replication in vivo, we substituted leucine or glycine for an active site methionine in yeast DNA polymerase ε (Pol ε). Compared to wild type Pol ε, ribonucleotide incorporation in vitro was 3-fold lower for M644L and 11-fold higher for M644G Pol ε. This hierarchy was re-capitulated in vivo in yeast strains lacking RNase H2. Moreover, the pol2-M644G rnh201Δ strain progressed more slowly through S-phase, had elevated dNTP pools and generated 2–5 base pair deletions in repetitive sequences at a high rate and gene orientation-dependent manner. The data indicate that ribonucleotides are incorporated during replication in vivo, that they are removed by RNase H2-dependent repair, and that defective repair results in replicative stress and genome instability via DNA strand misalignment.

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...

Data and materials availability: Antibody sequences have been deposited to GenBank under accession numbers MN643173 through MN643554. The cryo-EM maps and refined coordinates were deposited in the EMDB and RCSB PDB databases, respectively, under the following accession numbers: DH270 UCA (EMD-20817 and PDB ID 6UM5), DH270.6 (EMD-20818 and PDB ID 6UM6), and DH270.mu1(EMD-20819 and PDB ID 6UM7). The ARMADiLLO program is available for download at http://sites.duke.edu/ ARMADiLLO. All flow cytometry data are available upon request. All other data are in the main and supplementary figures and text.

Oner's (1971) computing expressions for nonparametric indices of sensitivity and bias are briefly reviewed, and shown to be incorrect when performance is below chance levels. Modifications of Grier's expressions, for cases when false alarms exceed hits, are presented, Pollack and Norman (1964) suggested general procedures for developing a nonparametric index of sensitivity, A', for recognition and detection experiments in which only a single data point is obtained in the unit square probability space representing hits and false alarms. Later Hodos (1970) suggested a nonparametric index of response bias, B", also based on the geometry of the square probability space. Finally, Grier (1971) derived explicit functional expressions for computing both of these indices. Grier's formulas have been widely used during the past 15 years, and have proved to be useful when the normal-distribution assumptions underlying traditional signal detection procedures fail, or when the assumptions are not testable.The application of Grier's (1971) formulas to detection data from individual subjects, some of whom perform below chance, can yield bizarre numbers. Some values of A', which should represent the area under an "averaged" ROC curve, can be negative rather than positive, and they can also be greater than 1.0 in absolute value. Further, values of B" for points to the left of the equal-bias diagonal can be negative rather than positive as they should be. The magnitudes of such erroneous A' and B" values can be large even when performance is only slightly below the chance diagonal. Discussions with colleagues suggested that some were unaware of the below-chance problem with Grier's formulas, and had reported values of A' and B* averaged over groups, without examining the individual subjects' data. If only a few subjects in a subset of conditions perform even slightly below chance, the averages would be distorted, but in ways that the researcher might not notice. Thus, we felt that it would be useful to provide a brief note on modifications of Grier's formulas that are appropriate for below-chance performance. Grier's FormulasSensitivity index. First, let us briefly review Grier's (1971) computing expressions. In Figure 1 -left, let y denote the proba-

SummaryThe impact of COVID-19 and the urgency to develop a vaccine against the SARS-CoV-2 virus cannot be overstated. The viral fusion spike (S) protein ectodomain is the primary target for vaccine development. Here we report an unexpected cold sensitivity of a stabilized SARS-CoV-2 ectodomain construct currently being widely used for immunogen design. We found that when stored at 22 or 37 °C for 1 week, the S-protein displayed well-ordered trimeric spikes by negative stain electron microscopy. However, storage at 4 °C reduced the trimeric spikes to <10%, accompanied by decreased stability and enhanced exposure of the ACE-2 receptor binding site. Well-formed S particles could be recovered from cold-stored samples by a brief incubation at 37 °C. Our results will have broad impact on structural, functional and vaccine studies using the SARS-CoV-2 S ectodomain.HighlightsSARS-CoV-2 S ectodomain construct, widely used for vaccine studies, exhibits cold sensitivity.Negative stain electron microscopy shows disintegration of spike structure upon storage at 4 °C.Differential scanning calorimetry measurements confirm destabilization by cold.Cold storage alters antigenicity of SARS-CoV-2 spike.Brief incubation at 37 °C restored spike integrity after cold-storage.

We have investigated the ability of the 3′ exonuclease activity of S. cerevisiae DNA polymerase ε (Pol ε) to proofread newly inserted ribonucleotides (rNMPs). During DNA synthesis in vitro, Pol ε 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 ε 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 ε does proofread newly inserted rNMPs to enhance genome stability.

The SARS-CoV-2 spike (S) protein, a primary target for COVID-19 vaccine development, presents its receptor binding domain in two conformations, the receptor-accessible 'up' or receptor-inaccessible 'down' states. Here we report that the commonly used stabilized S ectodomain construct '2P' is sensitive to cold temperatures, and this cold sensitivity is abrogated in a 'down' state-stabilized ectodomain. Our findings will impact structural, functional and vaccine studies that use the SARS-CoV-2 S ectodomain.The spike (S) protein of SARS-CoV-2 mediates receptor binding and cell entry and is a key target for vaccine development efforts. Stabilized S ectodomain constructs have been developed that mimic the native spike, bind the ACE-2 receptor 1,2 and present epitopes for neutralizing antibodies on their surface [3][4][5][6] . The so-called '2P' S ectodomain construct (2P S) comprises residues 1-1,208 of SARS-CoV-2 S and contains two proline (2P) substitutions in the C-terminal S2 domain designed to stabilize the prefusion S conformation; a C-terminal foldon trimerization motif and a mutation that abrogates the furin-cleavage site 1 (Fig. 1a). This and similar constructs have been widely used for structural biology and vaccine studies [1][2][3]7,8 . Purified S ectodomain proteins 9 are assessed for quality control by SDS-PAGE, size exclusion chromatography (SEC), differential scanning fluorimetry (DSF) 10 and negative-stain electron microscopy (NSEM). The last technique has been particularly informative because it reveals the structural integrity of individual molecules, allowing us to examine preparations that look similar by using bulk methods such as SDS-PAGE and SEC (Supplementary Fig. 1). The observed variability between preparations indicates a fragile S ectodomain, and measures to overcome the issue have been previously reported 11,12 .Here we link the apparent fragility of 2P S to its rapid denaturation on storage at 4 °C (Fig. 1b). We followed the structural, biophysical and antigenic properties of 2P S stored under different temperature conditions (Fig. 1, Extended Data Figs. 1 and 2, Supplementary Tables 1 and 2 and Supplementary Figs. 2-6). 2P S was produced in 293F cells at 37 °C and purified at room temperature within 6-8 h (Supplementary Fig. 1). We performed NSEM analysis of 2P S incubated at different temperatures (Fig. 1b and Extended Data Fig. 1d,e). Freshly prepared 2P S samples assessed on the same day they were purified showed on average 75% well-formed spikes, with characteristic kite-shaped morphology on NSEM micrographs

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