Rate Of Nucleotide Incorporation Research Articles

The enzymatic properties of Arabidopsis thaliana DNA polymerase λ suggest a role in base excision repair

Base excision repair (BER) generates gapped DNA intermediates containing a 5′-terminal 2-deoxyribose-5-phosphate (5′-dRP) group. In mammalian cells, gap filling and dRP removal are catalyzed by Pol β, which belongs to the X family of DNA polymerases. In higher plants, the only member of the X family of DNA polymerases is Pol λ. Although it is generally believed that plant Pol λ participates in BER, there is limited experimental evidence for this hypothesis. Here we have characterized the biochemical properties of Arabidopsis thaliana Pol λ (AtPol λ) in a BER context, using a variety of DNA repair intermediates. We have found that AtPol λ performs gap filling inserting the correct nucleotide, and that the rate of nucleotide incorporation is higher in substrates containing a C in the template strand. Gap filling catalyzed by AtPol λ is most efficient with a phosphate at the 5′-end of the gap and is not inhibited by the presence of a 5′-dRP mimic. We also show that AtPol λ possesses an intrinsic dRP lyase activity that is reduced by mutations at two lysine residues in its 8-kDa domain, one of which is present in Pol λ exclusively and not in any Pol β homolog. Importantly, we also found that the dRP lyase activity of AtPol λ allows efficient completion of uracil repair in a reconstituted short-patch BER reaction. These results suggest that AtPol λ plays an important role in plant BER.

Pre-Steady-State Kinetic Characterization of an Antibiotic-Resistant Mutant of Staphylococcus aureus DNA Polymerase PolC.

The emergence and spread of antibiotic resistance in bacterial pathogens are serious and ongoing threats to public health. Since chromosome replication is essential to cell growth and pathogenesis, the essential DNA polymerases in bacteria have long been targets of antimicrobial development, although none have yet advanced to the market. Here, we use transient-state kinetic methods to characterize the inhibition of the PolC replicative DNA polymerase from Staphylococcus aureus by 2-methoxyethyl-6-(3'-ethyl-4'-methylanilino)uracil (ME-EMAU), a member of the 6-anilinouracil compounds that specifically target PolC enzymes, which are found in low-GC content Gram-positive bacteria. We find that ME-EMAU binds to S. aureus PolC with a dissociation constant of 14 nM, more than 200-fold tighter than the previously reported inhibition constant, which was determined using steady-state kinetic methods. This tight binding is driven by a very slow off rate of 0.006 s-1. We also characterized the kinetics of nucleotide incorporation by PolC containing a mutation of phenylalanine 1261 to leucine (F1261L). The F1261L mutation decreases ME-EMAU binding affinity by at least 3,500-fold but also decreases the maximal rate of nucleotide incorporation by 11.5-fold. This suggests that bacteria acquiring this mutation would be likely to replicate slowly and be unable to out-compete wild-type strains in the absence of inhibitors, reducing the likelihood of the resistant bacteria propagating and spreading resistance.

Recording Temporal Signals with Minutes Resolution Using Enzymatic DNA Synthesis.

Employing DNA as a high-density data storage medium has paved the way for next-generation digital storage and biosensing technologies. However, the multipart architecture of current DNA-based recording techniques renders them inherently slow and incapable of recording fluctuating signals with subhour frequencies. To address this limitation, we developed a simplified system employing a single enzyme, terminal deoxynucleotidyl transferase (TdT), to transduce environmental signals into DNA. TdT adds nucleotides to the 3'-ends of single-stranded DNA (ssDNA) in a template-independent manner, selecting bases according to inherent preferences and environmental conditions. By characterizing TdT nucleotide selectivity under different conditions, we show that TdT can encode various physiologically relevant signals such as Co2+, Ca2+, and Zn2+ concentrations and temperature changes in vitro. Further, by considering the average rate of nucleotide incorporation, we show that the resulting ssDNA functions as a molecular ticker tape. With this method we accurately encode a temporal record of fluctuations in Co2+ concentration to within 1 min over a 60 min period. Finally, we engineer TdT to allosterically turn off in the presence of a physiologically relevant concentration of calcium. We use this engineered TdT in concert with a reference TdT to develop a two-polymerase system capable of recording a single-step change in the Ca2+ signal to within 1 min over a 60 min period. This work expands the repertoire of DNA-based recording techniques by developing a novel DNA synthesis-based system that can record temporal environmental signals into DNA with a resolution of minutes.

Rapid incorporation of Favipiravir by the fast and permissive viral RNA polymerase complex results in SARS-CoV-2 lethal mutagenesis

The ongoing Corona Virus Disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has emphasized the urgent need for antiviral therapeutics. The viral RNA-dependent-RNA-polymerase (RdRp) is a promising target with polymerase inhibitors successfully used for the treatment of several viral diseases. We demonstrate here that Favipiravir predominantly exerts an antiviral effect through lethal mutagenesis. The SARS-CoV RdRp complex is at least 10-fold more active than any other viral RdRp known. It possesses both unusually high nucleotide incorporation rates and high-error rates allowing facile insertion of Favipiravir into viral RNA, provoking C-to-U and G-to-A transitions in the already low cytosine content SARS-CoV-2 genome. The coronavirus RdRp complex represents an Achilles heel for SARS-CoV, supporting nucleoside analogues as promising candidates for the treatment of COVID-19.

Heterotrimeric PCNA increases the activity and fidelity of Dbh, a Y-family translesion DNA polymerase prone to creating single-base deletion mutations

Dbh is a Y-family translesion DNA polymerase from Sulfolobus acidocaldarius, an archaeal species that grows in harsh environmental conditions. Biochemically, Dbh displays a distinctive mutational profile, creating single-base deletion mutations at extraordinarily high frequencies (up to 50 %) in specific repeat sequences. In cells, however, Dbh does not appear to contribute significantly to spontaneous frameshifts in these same sequence contexts. This suggests that either the error-prone DNA synthesis activity of Dbh is reduced in vivo and/or Dbh is restricted from replicating these sequences. Here, we test the hypothesis that the propensity for Dbh to make single base deletion mutations is reduced through interaction with the S. acidocaldarius heterotrimeric sliding clamp processivity factor, PCNA-123. We first confirm that Dbh physically interacts with PCNA-123, with the interaction requiring both the PCNA-1 subunit and the C-terminal 10 amino acids of Dbh, which contain a predicted PCNA-interaction peptide (PIP) motif. This interaction stimulates the polymerase activity of Dbh, even on short, linear primer-template DNA, by increasing the rate of nucleotide incorporation. This stimulation requires an intact PCNA-123 heterotrimer and a DNA duplex length of at least 18 basepairs, the minimal length predicted from structural data to bind to both the polymerase and the clamp. Finally, we find that PCNA-123 increases the fidelity of Dbh on a single-base deletion hotspot sequence 3-fold by promoting an increase in the rate of correct, but not incorrect, nucleotide addition and propose that PCNA-123 induces Dbh to adopt a more active conformation that is less prone to creating deletions during DNA synthesis.

Defective Strand-Displacement DNA Synthesis Due to Accumulation of Thymidine Analogue Resistance Mutations in HIV-2 Reverse Transcriptase.

Retroviral reverse transcriptases (RTs) have the ability to carry out strand displacement DNA synthesis in the absence of accessory proteins. Although studies with RTs and other DNA polymerases suggest that fingers subdomain residues participate in strand displacement, molecular determinants of this activity are still unknown. A mutant human immunodeficiency virus type 2 (HIV-2) RT (M41L/D67N/K70R/S215Y) with low strand displacement activity was identified after screening a panel of purified enzymes, including several antiretroviral drug-resistant HIV-1 and HIV-2 RTs. In HIV-1, resistance to zidovudine and other thymidine analogues is conferred by different combinations of M41L, D67N, K70R, L210W, T215F/Y, and K219E/Q (designated as thymidine analogue resistance-associated mutations (TAMs)). However, those changes are rarely selected in HIV-2. We show that the strand displacement activity of HIV-2ROD mutants M41L/S215Y and D67N/K70R was only slightly reduced compared to the wild-type RT. In contrast, mutants D67N/K70R/S215Y and M41L/D67N/K70R/S215Y were the most defective RTs in reactions carried out with nicked and gapped substrates. Moreover, these enzymes showed the lowest nucleotide incorporation rates in assays carried out with strand displacement substrates. Unlike in HIV-2, substitutions M41L/T215Y and D67N/K70R/T215Y/K219Q had no effect on the strand displacement activity of HIV-1BH10 RT. The strand displacement efficiencies of HIV-2ROD RTs were consistent with the lower replication capacity of HIV-2 strains bearing the four major TAMs in their RT. Our results highlight the role of the fingers subdomain in strand displacement. These findings might be important for the development of strand-displacement defective RTs.

Rational Control of Poliovirus RNA-Dependent RNA Polymerase Fidelity by Modulating Motif-D Loop Conformational Dynamics.

The conserved structural motif D is an important determinant of the speed and fidelity of viral RNA-dependent RNA polymerases (RdRps). Structural and computational studies have suggested that conformational changes in the motif-D loop that help to reposition the catalytic lysine represent critical steps in nucleotide selection and incorporation. Conformations of the motif-D loop in the poliovirus RdRp are likely controlled in part by noncovalent interactions involving the motif-D residue Glu364. This residue swivels between making interactions with Lys228 and Asn370 to stabilize the open and closed loop conformations, respectively. We show here that we can rationally control the motif-D loop conformation by breaking these interactions. The K228A variant favors a more active closed conformation, leading to increased nucleotide incorporation rates and decreased nucleotide selectivity, and the N370A variant favors a less active open conformation, leading to decreased nucleotide incorporation rates and increased nucleotide selectivity. Similar competing interactions likely control nucleotide incorporation rates and fidelity in other viral RdRps. Rational engineering of these interactions may be important in the generation of live, attenuated vaccine strains, considering the established relationships between RdRp function and viral pathogenesis.

Structure of HIV-1 RT/dsRNA initiation complex prior to nucleotide incorporation

The initiation phase of HIV reverse transcription has features that are distinct from its elongation phase. The first structure of a reverse transcription initiation complex (RTIC) that trapped the complex after incorporation of one ddCMP nucleotide was published recently [Larsen KP, et al. (2018) Nature 557:118-122]. Here we report a crystal structure of a catalytically active HIV-1 RT/dsRNA complex that mimics the state of the RTIC before the first nucleotide incorporation. The structure reveals that the dsRNA-bound conformation of RT is closer to that of RT bound to a nonnucleoside RT inhibitor (NNRTI) and dsDNA; a hyperextended thumb conformation helps to accommodate the relatively wide dsRNA duplex. The RNA primer 3' end is positioned 5 Å away from the polymerase site; however, unlike in an NNRTI-bound state in which structural elements of RT restrict the movement of the primer, the primer terminus of dsRNA is not blocked from reaching the active site of RT. The observed structural changes and energetic cost of bringing the primer 3' end to the priming site are hypothesized to explain the slower nucleotide incorporation rate of the RTIC. An unusual crystal lattice interaction of dsRNA with its symmetry mate is reminiscent of the RNA architecture within the extended vRNA-tRNALys3 in the RTIC. This RT/dsRNA complex captures the key structural characteristics and components of the RTIC, including the RT conformational changes and interactions with the dsRNA primer-binding site region, and these features have implications for better understanding of RT initiation.

PCNA accelerates the nucleotide incorporation rate by DNA polymerase δ

DNA polymerase delta (Pol δ) is responsible for the elongation and maturation of Okazaki fragments in eukaryotic cells. Proliferating cell nuclear antigen (PCNA) recruits Pol δ to the DNA and serves as a processivity factor. Here, we show that PCNA also stimulates the catalytic rate of Saccharomyces cerevisiae Pol δ by >10-fold. We determined template/primer DNA binding affinities and stoichiometries by Pol δ in the absence of PCNA, using electrophoretic mobility shift assays, fluorescence intensity changes and fluorescence anisotropy binding titrations. We provide evidence that Pol δ forms higher ordered complexes upon binding to DNA. The Pol δ catalytic rates in the absence and presence of PCNA were determined at millisecond time resolution using quench flow kinetic measurements. The observed rate for single nucleotide incorporation by a preformed DNA-Pol δ complex in the absence of PCNA was 40 s−1. PCNA enhanced the nucleotide incorporation rate by >10 fold. Compared to wild-type, a growth-defective yeast PCNA mutant (DD41,42AA) showed substantially less stimulation of the Pol δ nucleotide incorporation rate, identifying the face of PCNA that is important for the acceleration of catalysis.

NETSeq reveals heterogeneous nucleotide incorporation by RNA polymerase I

DNA sequence motifs that affect RNA polymerase transcription elongation are well studied in prokaryotic organisms and contribute directly to regulation of gene expression. Despite significant work on the regulation of eukaryotic transcription, the effect of DNA template sequence on RNA polymerase I (Pol I) transcription elongation remains unknown. In this study, we examined the effects of DNA sequence motifs on Pol I transcription elongation kinetics in vitro and in vivo. Specifically, we characterized how the spy rho-independent terminator motif from Escherichia coli directly affects Saccharomyces cerevisiae Pol I activity, demonstrating evolutionary conservation of sequence-specific effects on transcription. The insight gained from this analysis led to the identification of a homologous sequence in the ribosomal DNA of S. cerevisiae We then used native elongating transcript sequencing (NETSeq) to determine whether Pol I encounters pause-inducing sequences in vivo. We found hundreds of positions within the ribosomal DNA (rDNA) that reproducibly induce pausing in vivo. We also observed significantly lower Pol I occupancy at G residues in the rDNA, independent of other sequence context, indicating differential nucleotide incorporation rates for Pol I in vivo. These data demonstrate that DNA template sequence elements directly influence Pol I transcription elongation. Furthermore, we have developed the necessary experimental and analytical methods to investigate these perturbations in living cells going forward.

Catalytic properties of RNA polymerases IV and V: accuracy, nucleotide incorporation and rNTP/dNTP discrimination.

All eukaryotes have three essential nuclear multisubunit RNA polymerases, abbreviated as Pol I, Pol II and Pol III. Plants are remarkable in having two additional multisubunit RNA polymerases, Pol IV and Pol V, which synthesize noncoding RNAs that coordinate RNA-directed DNA methylation for silencing of transposons and a subset of genes. Based on their subunit compositions, Pols IV and V clearly evolved as specialized forms of Pol II, but their catalytic properties remain undefined. Here, we show that Pols IV and V differ from one another, and Pol II, in nucleotide incorporation rate, transcriptional accuracy and the ability to discriminate between ribonucleotides and deoxyribonucleotides. Pol IV transcription is considerably more error-prone than Pols II or V, which may be tolerable in its synthesis of short RNAs that serve as precursors for siRNAs targeting non-identical members of transposon families. By contrast, Pol V exhibits high fidelity transcription, similar to Pol II, suggesting a need for Pol V transcripts to faithfully reflect the DNA sequence of target loci to which siRNA–Argonaute silencing complexes are recruited.

HIV-1 Reverse Transcriptase Polymerase and RNase H (Ribonuclease H) Active Sites Work Simultaneously and Independently

HIV reverse transcriptase plays a central role in viral replication and requires coordination of both polymerase and RNase H activities. Although this coordination is crucial in viral replication, whether a DNA/RNA hybrid can simultaneously engage both active sites has yet to be determined as structural and kinetic analyses have provided contradictory results. Single nucleotide incorporation and RNase H cleavage were examined using presteady-state kinetics with global data analysis. The results revealed three interconverting reverse transcriptase-DNA/RNA species; 43% were active for both sites simultaneously, 27% showed only polymerase activity, and the remaining 30% were nonproductive. Our data clearly demonstrated that the DNA/RNA hybrid could contact both active sites simultaneously, although the single nucleotide incorporation (105 s-1) was ∼5-fold faster than the cleavage (23 s-1). By using a series of primers with different lengths, we found that a string of at least 4-6 nucleotides downstream of the cleaving site was required for efficient RNA cleavage. This was corroborated by our observations that during processive nucleotide incorporation, sequential rounds of RNA cleavage occurred each time after ∼6 nucleotides were incorporated. More importantly, during processive primer extension, pyrophosphate (PPi) release was rate-limiting so that the average rate of nucleotide incorporation (∼28 s-1) was comparable with that of net RNA cleavage (∼27 nucleotides(s)). Although polymerization is efficient and processive, RNase H is inefficient and periodic. This combination allows the two catalytic centers of HIVRT to work simultaneously at similar speeds without being tightly coupled.

TSC loss distorts DNA replication programme and sensitises cells to genotoxic stress.

Tuberous Sclerosis (TSC) is characterized by exorbitant mTORC1 signalling and manifests as non-malignant, apoptosis-prone neoplasia. Previous reports have shown that TSC-/- cells are highly susceptible to mild, innocuous doses of genotoxic stress, which drive TSC-/- cells into apoptotic death. It has been argued that this hypersensitivity to stress derives from a metabolic/energetic shortfall in TSC-/- cells, but how metabolic dysregulation affects the DNA damage response and cell cycle alterations in TSC-/- cells exposed to genotoxic stress is not understood. We report here the occurrence of futile checkpoint responses and an unusual type of replicative stress (RS) in TSC1-/- fibroblasts exposed to low-dose genotoxins. This RS is characterized by elevated nucleotide incorporation rates despite only modest origin over-firing. Strikingly, an increased propensity for asymmetric fork progression and profuse chromosomal aberrations upon mild DNA damage confirmed that TSC loss indeed proved detrimental to stress adaptation. We conclude that low stress tolerance of TSC-/- cells manifests at the level of DNA replication control, imposing strong negative selection on genomic instability that could in turn detain TSC-mutant tumours benign.

Investigation of Intradomain Motions of a Y-Family DNA Polymerase during Substrate Binding and Catalysis.

DNA polymerases catalyze DNA synthesis through a stepwise kinetic mechanism that begins with binding to DNA, followed by selection, binding, and incorporation of a nucleotide into an elongating primer. It is hypothesized that subtle active site adjustments in a polymerase to align reactive moieties limit the rate of correct nucleotide incorporation. DNA damage can impede this process for many DNA polymerases, causing replication fork stalling, genetic mutations, and potentially cell death. However, specialized Y-family DNA polymerases are structurally evolved to efficiently bypass DNA damage in vivo, albeit at the expense of replication fidelity. Dpo4, a model Y-family polymerase from Sulfolobus solfataricus, has been well-studied kinetically, structurally, and computationally, which yielded a mechanistic understanding of how the Y-family DNA polymerases achieve their unique catalytic properties. We previously employed a real-time Förster resonance energy transfer (FRET) technique to characterize the global conformational motions of Dpo4 during DNA binding as well as nucleotide binding and incorporation by monitoring changes in distance between sites on the polymerase and DNA, and even between domains of Dpo4. Here, we extend the utility of our FRET methodology to observe conformational transitions within individual domains of Dpo4 during DNA binding and nucleotide incorporation. The results of this novel, intradomain FRET approach unify findings from many studies to fully clarify the complex DNA binding mechanism of Dpo4. Furthermore, intradomain motions in the Finger domain during nucleotide binding and incorporation, for the first time, report on the rate-limiting step of a single-nucleotide addition catalyzed by Dpo4.

Abstract 4645: Cell cycle specific effects and associated DNA damage of selective inhibitors of nuclear export (SINE)

Abstract Nuclear export of proteins is fundamental for cell growth and function. Selinexor is a SINE compound that is in clinical development for the treatment of different cancers. Selinexor forms a slowly reversible covalent bond to Exportin-1 (XPO1), preventing its association with protein cargos, thereby resulting in their nuclear retention. XPO1 cargos include the majority of tumor suppressor proteins (TSP) and cell cycle regulators such as p53, p21 and p27 that have key roles in cancer progression and drug response. It is unclear how selinexor affects cell cycle progression in individual cells and the subsequent stress and fate of those cells. To elucidate selinexor action, we developed cell lines that stably express fluorescent ubiquitin cell cycle reporters (FUCCI), and followed individual cells longitudinally using continuous time-lapse microscopy for 72 hours. We report that in fibrosarcoma-derived HT-1080 cells that express wildtype p53 and p21, 18% of the initial cell population became arrested with >90% in G1- or S-phase and 40% died with 64% from G1- or early S-phase after a cell cycle delay or arrest. We also found that 42% of cells divided, but the progeny died or arrested in G1- or S-phase of the next cell cycle - often after cell cycle arrest or slowed cell cycle progression. Using FUCCI, we tracked the response of cells treated acutely in specific cell cycle stages. Cells treated in G1-phase most often arrested or died in G1- or S-phase, whereas cells treated in G2-phase usually progressed to cell division. As FUCCI revealed S-phase progression defects and associated cell death, we further characterized this phenotype. Using nucleotide incorporation with fluorescent detection, we observed that as early as 2 hours after selinexor treatment only a subset of cells is undergoing DNA replication. Cells that were able to replicate their DNA are doing that inefficiently as both the rate and maximal levels of nucleotide incorporation are significantly reduced. S-phase arrest and progression defects may manifest as DNA double-strand breaks. We find a strong association between S-phase status and DNA damage. In some cells, the damage occurs within hours of selinexor treatment and appears as a striking cluster of foci. At 8 hours, nearly 35-45% of cells contain DNA damage clusters. Importantly, the damage clusters sometimes repair as determined by fixed cell time-course analysis and live-cell microscopy. We are exploring the nature of these DNA damage structures and the mechanisms of their formation and repair. In summary, selinexor is fast acting, shows cell cycle selectivity, results in DNA damage and is highly effective at arresting cell growth and inducing apoptosis in tumor cells. These data suggest that selinexor may exert anti-cancer effects even on slow growing tumors where the bulk of the cell mass presents a G1-like state and that it likely combines well with other cell cycle targeted therapeutics. Citation Format: Russell T. Burke, Joshua Marcus, John DeSisto, Yosef Landesman, James D. Orth. Cell cycle specific effects and associated DNA damage of selective inhibitors of nuclear export (SINE). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4645.

Activation of the Erythroid Transcriptional Program in Murine Adult Bone Marrow Takes Place during a Faster, Shorter S Phase and Is Dependent on S Phase Progression

We previously characterized an S phase-dependent commitment switch in mouse fetal liver. Specifically, the upregulation of CD71 in the transition from Subset 0 (S0, Lin-CD71medium) to Subset 1 (S1, Lin-CD71high) identifies cells in early S phase of the last colony-forming-unit-erythroid (CFUe) generation, undergoing key differentiation events, including onset of Erythropoietin (Epo) dependence, GATA-1 activation and reconfiguration of chromatin at the §-globin locus control region. This commitment switch requires S phase progression, and is regulated by the S-phase- dependent downregulation of the transcription factor PU.1, a GATA-1 antagonist. Of note, the specific cell cycle S phase in which this commitment switch takes place differs from S phase in previous cycles, in that it is shorter, with a 50% faster rate of nucleotide incorporation into DNA.Here, we investigate whether a similar commitment switch takes place in adult bone-marrow. Using fresh bone-marrow from mice in the basal state or following Epo injection, we determined colony-forming potential, transcriptional profiles and cell cycle status of Kit+Lin- subsets defined by cell surface markers that have been previously implicated as enriched in the megakaryocytic-erythrocytic lineages: CD105, CD150, CD55, CD71, in conjunction with a PU.1-GFP reporter mouse.Using quantitative RT-PCR together with flow-cytometric analysis and the SPADE algorithm we identified a linear erythroid developmental sequence of Epo-responsive Kit+Lin-CD55+ bone-marrow subsets, which gradually declined in PU.1, transiently increased CD150, and upregulated both CD105 and CD71. The loss of PU.1 coincided with increased GATA1 levels and the transcriptional activation of erythroid genes. CFUe activity peaked within Kit+Lin-CD55+ cells that were also CD105+, where colonies of other lineage potential were rare (Fig 1).Upregulation of CD71 in erythroid cells indicates the onset of Epo receptor signaling and Epo dependence. We found that in Kit+Lin-CD55+ bone-marrow in vivo, high levels of CD71 were highly correlated with S phase (Fig 2). Furthermore, the rate of incorporation of the nucleotide analogue BrdU was substantially higher in CD71 -high cells, suggesting a rapid, short S phase. To functionally examine the role of S phase, we isolated Kit+Lin-CD55+CD105+ cells that were CD71 low, and followed their differentiation in vitro. These cells upregulate CD71 within 10 to 16 hours, followed by upregulation of Ter119. Upregulation of CD71 was Epo dependent, and took place in S phase; further, it was reversibly inhibited by the DNA polymerase inhibitor, Aphidicolin, suggesting dependence on S phase progression.We conclude that adult bone marrow Kit+Lin-CD55+CD105+ are at the CFUe developmental stage, and undergo an Epo and S-phase dependent commitment switch that activates the erythroid transcriptional and differentiation program. S phase during this commitment switch is characterized by a fast DNA synthesis rate. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Nucleobase but not Sugar Fidelity is Maintained in the Sabin I RNA-Dependent RNA Polymerase.

The Sabin I poliovirus live, attenuated vaccine strain encodes for four amino acid changes (i.e., D53N, Y73H, K250E, and T362I) in the RNA-dependent RNA polymerase (RdRp). We have previously shown that the T362I substitution leads to a lower fidelity RdRp, and viruses encoding this variant are attenuated in a mouse model of poliovirus. Given these results, it was surprising that the nucleotide incorporation rate and nucleobase fidelity of the Sabin I RdRp is similar to that of wild-type enzyme, although the Sabin I RdRp is less selective against nucleotides with modified sugar groups. We suggest that the other Sabin amino acid changes (i.e., D53N, Y73H, K250E) help to re-establish nucleotide incorporation rates and nucleotide discrimination near wild-type levels, which may be a requirement for the propagation of the virus and its efficacy as a vaccine strain. These results also suggest that the nucleobase fidelity of the Sabin I RdRp likely does not contribute to viral attenuation.

Abstract 1760: Single cell longitudinal studies reveal cell cycle specific effects of selective inhibitors of nuclear export (SINE)

Abstract Nuclear export of proteins is fundamental for cell growth and function. Selinexor is a SINE compound that is in clinical development for the treatment of different cancers. Selinexor forms a slowly reversible covalent bond to Exportin-1 (XPO1), preventing its association with protein cargos, thereby resulting in their nuclear retention. XPO1 cargos include the majority of tumor suppressor proteins (TSP) and cell cycle regulators such as p53, p21 and p27 that have key roles in cancer progression and drug response. It is unclear how selinexor affects cell cycle progression in individual cells and the subsequent stress and fate of those cells. To elucidate selinexor action, we developed cell lines that stably express fluorescent ubiquitin cell cycle reporters (FUCCI), and followed individual cells longitudinally using continuous time-lapse microscopy for 72 hours. We report that in fibrosarcoma-derived HT-1080 cells that express wildtype p53 and p21, 20% of the initial cell population became arrested with >90% in G1- or S-phase and 35% died with nearly 70% from G1- or S-phase after a cell cycle delay or arrest. We also found that 45% of cells divided, but the progeny died or arrested in G1- or S-phase of the next cell cycle - often after cell cycle arrest or slowed cell cycle progression. Using FUCCI, we tracked the response of cells treated acutely in specific cell cycle stages. Cells treated in G1-phase most often arrested or died in G1- or S-phase, whereas cells treated in G2-phase usually progressed to cell division. As FUCCI revealed S-phase progression defects and associated cell death, we further characterized this phenotype. Using nucleotide incorporation with fluorescent detection, we find that as soon as 8 hours after selinexor fewer cells are undergoing DNA replication and those that are, are doing so inefficiently as both the rate and maximal levels of nucleotide incorporation are significantly reduced. S-phase arrest and progression defects may manifest as DNA double-strand breaks. We find a strong association between S-phase status and DNA damage. In some cells, the damage occurs within hours of selinexor treatment and appears as a striking cluster of foci. At 8 hours, nearly 50% of cells contain DNA damage clusters. Importantly, the damage clusters sometimes repair as determined by fixed cell time-course analysis and live-cell microscopy. We are exploring the nature of these DNA damage structures, the mechanisms of their formation and repair, and are also testing for redundancy of the cell cycle regulators in determining selinexor responses. In summary, selinexor is fast acting, shows cell cycle selectivity, and is highly effective at arresting cell growth and inducing apoptosis in tumor cells. These data suggest that selinexor may exert anti-cancer effects even on slow growing tumors where the bulk of the cell mass presents a G1-like state and that it likely combines well with other cell cycle targeted therapeutics. Citation Format: Russell T. Burke, Joshua M. Marcus, John DeSisto, Yosef Landesman, James D. Orth. Single cell longitudinal studies reveal cell cycle specific effects of selective inhibitors of nuclear export (SINE). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1760. doi:10.1158/1538-7445.AM2015-1760

Significant contribution of the 3'→5' exonuclease activity to the high fidelity of nucleotide incorporation catalyzed by human DNA polymerase ϵ.

Most eukaryotic DNA replication is performed by A- and B-family DNA polymerases which possess a faithful polymerase activity that preferentially incorporates correct over incorrect nucleotides. Additionally, many replicative polymerases have an efficient 3′→5′ exonuclease activity that excises misincorporated nucleotides. Together, these activities contribute to overall low polymerase error frequency (one error per 106–108 incorporations) and support faithful eukaryotic genome replication. Eukaryotic DNA polymerase ϵ (Polϵ) is one of three main replicative DNA polymerases for nuclear genomic replication and is responsible for leading strand synthesis. Here, we employed pre-steady-state kinetic methods and determined the overall fidelity of human Polϵ (hPolϵ) by measuring the individual contributions of its polymerase and 3′→5′ exonuclease activities. The polymerase activity of hPolϵ has a high base substitution fidelity (10−4–10−7) resulting from large decreases in both nucleotide incorporation rate constants and ground-state binding affinities for incorrect relative to correct nucleotides. The 3′→5′ exonuclease activity of hPolϵ further enhances polymerization fidelity by an unprecedented 3.5 × 102 to 1.2 × 104-fold. The resulting overall fidelity of hPolϵ (10−6–10−11) justifies hPolϵ to be a primary enzyme to replicate human nuclear genome (0.1–1.0 error per round). Consistently, somatic mutations in hPolϵ, which decrease its exonuclease activity, are connected with mutator phenotypes and cancer formation.

Sphingosine, a Modulator of Human Translesion DNA Polymerase Activity

Translesion (TLS) DNA polymerases are specialized, error-prone enzymes that synthesize DNA across bulky, replication-stalling DNA adducts. In so doing, they facilitate the progression of DNA synthesis and promote cell proliferation. To potentiate the effect of cancer chemotherapeutic regimens, we sought to identify inhibitors of TLS DNA polymerases. We screened five libraries of ∼ 3000 small molecules, including one comprising ∼ 600 nucleoside analogs, for their effect on primer extension activity of DNA polymerase η (Pol η). We serendipitously identified sphingosine, a lipid-signaling molecule that robustly stimulates the activity of Pol η by ∼ 100-fold at low micromolar concentrations but inhibits it at higher concentrations. This effect is specific to the Y-family DNA polymerases, Pols η, κ, and ι. The addition of a single phosphate group on sphingosine completely abrogates this effect. Likewise, the inclusion of other sphingolipids, including ceramide and sphingomyelin to extension reactions does not elicit this response. Sphingosine increases the rate of correct and incorrect nucleotide incorporation while having no effect on polymerase processivity. Endogenous Pol η activity is modulated similarly as the recombinant enzyme. Importantly, sphingosine-treated cells exhibit increased lesion bypass activity, and sphingosine tethered to membrane lipids mimics the effects of free sphingosine. Our studies have uncovered sphingosine as a modulator of TLS DNA polymerase activity; this property of sphingosine may be associated with its known role as a signaling molecule in regulating cell proliferation in response to cellular stress.