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We present avidity sequencing, a sequencing chemistry that separately optimizes the processes of stepping along a DNA template and that of identifying each nucleotide within the template. Nucleotide identification uses multivalent nucleotide ligands on dye-labeled cores to form polymerase–polymer–nucleotide complexes bound to clonal copies of DNA targets. These polymer–nucleotide substrates, termed avidites, decrease the required concentration of reporting nucleotides from micromolar to nanomolar and yield negligible dissociation rates. Avidity sequencing achieves high accuracy, with 96.2% and 85.4% of base calls having an average of one error per 1,000 and 10,000 base pairs, respectively. We show that the average error rate of avidity sequencing remained stable following a long homopolymer.

We present avidity sequencing - a novel sequencing chemistry that separately optimizes the process of stepping along a DNA template and the process of identifying each nucleotide within the template. Nucleotide identification uses multivalent nucleotide ligands on dye-labeled cores to form polymerase-polymer nucleotide complexes bound to clonal copies of DNA targets. These polymer-nucleotide substrates, termed avidites, decrease the required concentration of reporting nucleotides from micromolar to nanomolar, and yield negligible dissociation rates. We demonstrate the use of avidites as a key component of a sequencing technology that surpasses Q40 accuracy and enables a diversity of applications that include single cell RNA-seq and whole human genome sequencing. We also show the advantages of this technology in sequencing through long homopolymers.

New atomic-resolution structures provide detailed insights into the molecular mechanism of telomerase, ushering in a new era of rational drug design to target telomerase in human genetic disorders and cancer.

Telomeres safeguard the genome by suppressing illicit DNA damage responses at chromosome termini. In order to compensate for incomplete DNA replication at telomeres, most continually dividing cells, including many cancers, express the telomerase ribonucleoprotein (RNP) complex. Telomerase maintains telomere length by catalyzing de novo synthesis of short DNA repeats using an internal telomerase RNA (TR) template. TRs from diverse species harbor structurally conserved domains that contribute to RNP biogenesis and function. In vertebrate TRs, the conserved regions 4 and 5 (CR4/5) fold into a three-way junction (3WJ) that binds directly to the telomerase catalytic protein subunit and is required for telomerase function. We have analyzed the structural properties of the human TR (hTR) CR4/5 domain using a combination of in vitro chemical mapping, endogenous RNP assembly assays, and single-molecule structural analysis. Our data suggest that a functionally essential stem loop within CR4/5 is not stably folded in the absence of the telomerase reverse transcriptase protein subunit in vitro. Rather, the hTR CR4/5 domain adopts a heterogeneous ensemble of conformations. RNA structural engineering intended to bias the folding landscape of the hTR CR4/5 demonstrates that a stably folded 3WJ motif is necessary but not sufficient to promote assembly of a functional RNP complex. Finally, single-molecule measurements on the hTR CR4/5 domain show that RNP assembly selects for a conformation that is not the major population in the heterogeneous free RNA ensemble, suggesting that non-canonical hTR folds may be required during telomerase biogenesis.

Telomerase reverse transcribes short guanine (G)-rich DNA repeat sequences from its internal RNA template to maintain telomere length. G-rich telomere DNA repeats readily fold into G-quadruplex (GQ) structures in vitro, and the presence of GQ-prone sequences throughout the genome introduces challenges to replication in vivo. Using a combination of ensemble and single-molecule telomerase assays, we discovered that GQ folding of the nascent DNA product during processive addition of multiple telomere repeats modulates the kinetics of telomerase catalysis and dissociation. Telomerase reactions performed with telomere DNA primers of varying sequence or using GQ-stabilizing K(+) versus GQ-destabilizing Li(+) salts yielded changes in DNA product profiles consistent with formation of GQ structures within the telomerase–DNA complex. Addition of the telomerase processivity factor POT1–TPP1 altered the DNA product profile, but was not sufficient to recover full activity in the presence of Li(+) cations. This result suggests GQ folding synergizes with POT1–TPP1 to support telomerase function. Single-molecule Förster resonance energy transfer experiments reveal complex DNA structural dynamics during real-time catalysis in the presence of K(+) but not Li(+), supporting the notion of nascent product folding within the active telomerase complex. To explain the observed distributions of telomere products, we globally fit telomerase time-series data to a kinetic model that converges to a set of rate constants describing each successive telomere repeat addition cycle. Our results highlight the potential influence of the intrinsic folding properties of telomere DNA during telomerase catalysis, and provide a detailed characterization of GQ modulation of polymerase function.

Telomerase maintains telomere length by reverse transcribing short G-rich DNA repeat sequences from its internal RNA template. G-rich telomere DNA repeats readily fold into G-quadruplex (GQ) structures in vitro, and the presence of GQ-prone sequences throughout the genome introduces challenges to replication in vivo. Using a combination of ensemble and single-molecule telomerase assays we discovered that GQ folding of the nascent DNA product during processive addition of multiple telomere repeats modulates the kinetics of telomerase catalysis and dissociation. Telomerase reactions performed with telomere DNA primers of varying sequence or using K+ versus Li+ salts yield changes in DNA product profiles consistent with formation of GQ structure within the telomerase-DNA complex. Single-molecule FRET experiments reveal complex DNA structural dynamics during real-time catalysis, supporting the notion of nascent product folding within the active telomerase complex. To explain the observed distributions of telomere products, we fit telomerase time series data to a global kinetic model that converges to a unique set of rate constants describing each successive telomere repeat addition cycle. Our results highlight the potential influence of the intrinsic folding properties of telomere DNA during telomerase catalysis and provide a detailed characterization of GQ modulation of polymerase function.SIGNIFICANCETelomeres protect the ends of linear chromosomes from illicit DNA processing events that can threaten genome stability. Telomere structure is built upon repetitive G-rich DNA repeat sequences that have the ability to fold into stable secondary structures called G-quadruplexes (GQs). In rapidly dividing cells, including the majority of human cancers, telomeres are maintained by the specialized telomerase enzyme. Thus, telomerase and its telomere DNA substrates represent important targets for developing novel cancer drugs. In this work, we provide evidence for GQ folding within the newly synthesized DNA product of an actively extending telomerase enzyme. Our results highlight the delicate interplay between the structural properties of telomere DNA and telomerase function.

Cell Reports, 20 (1), ISSN:2666-3864, ISSN:2211-1247

The TRAMP complex is involved in the nuclear surveillance and turnover of noncoding RNAs and intergenic transcripts. TRAMP is associated with the nuclear exosome and consists of a poly(A)polymerase subcomplex (Trf4-Air2) and a helicase (Mtr4). We found that N-terminal low-complexity regions of Trf4 and Air2 bind Mtr4 in a cooperative manner. The 2.4 Å resolution crystal structure of the corresponding ternary complex reveals how Trf4 and Air2 wrap around the DExH core of the helicase. Structure-based mutations on the DExH core impair binding to Trf4 and Air2, and also to Trf5 and Air1. The combination of structural, biochemical, and biophysical data suggests that the poly(A)polymerase core of Trf4-Air2 is positioned below the base of the helicase, where the unwound 3' end of an RNA substrate is expected to emerge. The results reveal conceptual similarities between the two major regulators of the exosome, the nuclear TRAMP and cytoplasmic Ski complexes.

Mtr4 is a conserved RNA helicase that functions together with the nuclear exosome. It participates in the processing of structured RNAs, including the maturation of 5.8S ribosomal RNA (rRNA). It also interacts with the polyadenylating Trf4-Air2 heterodimer to form the so-called TRAMP ( Tr f4- A ir2- M tr4 P olyadenylation) complex. TRAMP is involved in exosome-mediated degradation of aberrant RNAs in nuclear surveillance pathways. We report the 2.9-Å resolution crystal structure of Saccharomyces cerevisiae Mtr4 in complex with ADP and RNA. The structure shows a central ATPase core similar to that of other DExH helicases. Inserted in the DExH core is a region characteristic of Mtr4 orthologues that folds into an elongated stalk connected to a β-barrel domain. This domain shows unexpected similarity to the KOW domain of L24, a ribosomal protein that binds 23S rRNA. We find that indeed the KOW domain of Mtr4 is able to bind in vitro transcribed tRNA iMet , suggesting it might assist in presenting RNA substrates to the helicase core. The interaction of Mtr4 with Trf4-Air2 is mediated not by the stalk/KOW insertion but by the DExH core. We find that in the context of the TRAMP complex, the DExH core functions independently in vitro as an RNA helicase and a protein-binding platform. Mtr4 has thus evolved specific structural and surface features to perform its multiple functions.

Cell 139, 1084-1095 (2009). doi:10.1016/j.cell.2009.11.015, Published by Cell Press, [Cambridge, Mass.]

Ribosome-associated complex (RAC) consists of the Hsp40 homolog Zuo1 and the Hsp70 homolog Ssz1. The chaperone participates in the biogenesis of newly synthesized polypeptides. Here we have identified yeast Rpl31, a component of the large ribosomal subunit, as a contact point of RAC at the polypeptide tunnel exit. Rpl31 is encoded by RPL31a and RPL31b, two closely related genes. Delta rpl31a Delta rpl31b displayed slow growth and sensitivity to low as well as high temperatures. In addition, Delta rpl31a Delta rpl31b was highly sensitive toward aminoglycoside antibiotics and suffered from defects in translational fidelity. With the exception of sensitivity at elevated temperature, the phenotype resembled yeast strains lacking one of the RAC subunits or Rpl39, another protein localized at the tunnel exit. Defects of Delta rpl31a Delta rpl31b Delta zuo1 did not exceed that of Delta rpl31a Delta rpl31b or Delta zuo1. However, the combined deletion of RPL31a, RPL31b, and RPL39 was lethal. Moreover, RPL39 was a multicopy suppressor, whereas overexpression of RAC failed to rescue growth defects of Delta rpl31a Delta rpl31b. The findings are consistent with a model in that Rpl31 and Rpl39 independently affect a common ribosome function, whereas Rpl31 and RAC are functionally interdependent. Rpl31, while not essential for binding of RAC to the ribosome, might be involved in proper function of the chaperone complex.