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The new coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses an RNA-dependent RNA polymerase (RdRp) for the replication of its genome and the transcription of its genes 1 – 3 . Here we present a cryo-electron microscopy structure of the SARS-CoV-2 RdRp in an active form that mimics the replicating enzyme. The structure comprises the viral proteins non-structural protein 12 (nsp12), nsp8 and nsp7, and more than two turns of RNA template–product duplex. The active-site cleft of nsp12 binds to the first turn of RNA and mediates RdRp activity with conserved residues. Two copies of nsp8 bind to opposite sides of the cleft and position the second turn of RNA. Long helical extensions in nsp8 protrude along exiting RNA, forming positively charged ‘sliding poles’. These sliding poles can account for the known processivity of RdRp that is required for replicating the long genome of coronaviruses 3 . Our results enable a detailed analysis of the inhibitory mechanisms that underlie the antiviral activity of substances such as remdesivir, a drug for the treatment of coronavirus disease 2019 (COVID-19) 4 . A cryo-electron microscopy structure of the RNA-dependent RNA polymerase of SARS-CoV-2 sheds light on coronavirus replication and enables the analysis of the inhibitory mechanisms of candidate antiviral drugs.

The synthesis of pre-mRNA by RNA polymerase II (Pol II) involves the formation of a transcription initiation complex, and a transition to an elongation complex 1 – 4 . The large subunit of Pol II contains an intrinsically disordered C-terminal domain that is phosphorylated by cyclin-dependent kinases during the transition from initiation to elongation, thus influencing the interaction of the C-terminal domain with different components of the initiation or the RNA-splicing apparatus 5 , 6 . Recent observations suggest that this model provides only a partial picture of the effects of phosphorylation of the C-terminal domain 7 – 12 . Both the transcription-initiation machinery and the splicing machinery can form phase-separated condensates that contain large numbers of component molecules: hundreds of molecules of Pol II and mediator are concentrated in condensates at super-enhancers 7 , 8 , and large numbers of splicing factors are concentrated in nuclear speckles, some of which occur at highly active transcription sites 9 – 12 . Here we investigate whether the phosphorylation of the Pol II C-terminal domain regulates the incorporation of Pol II into phase-separated condensates that are associated with transcription initiation and splicing. We find that the hypophosphorylated C-terminal domain of Pol II is incorporated into mediator condensates and that phosphorylation by regulatory cyclin-dependent kinases reduces this incorporation. We also find that the hyperphosphorylated C-terminal domain is preferentially incorporated into condensates that are formed by splicing factors. These results suggest that phosphorylation of the Pol II C-terminal domain drives an exchange from condensates that are involved in transcription initiation to those that are involved in RNA processing, and implicates phosphorylation as a mechanism that regulates condensate preference. RNA polymerase II with a hypophosphorylated C-terminal domain preferentially incorporates into mediator condensates, and with a hyperphosphorylated C-terminal domain into splicing-factor condensates, revealing phosphorylation as a regulatory mechanism in condensate preference.

Respiratory syncytial virus (RSV) and human metapneumovirus (HMPV) cause severe respiratory diseases in infants and elderly adults 1 . No vaccine or effective antiviral therapy currently exists to control RSV or HMPV infections. During viral genome replication and transcription, the tetrameric phosphoprotein P serves as a crucial adaptor between the ribonucleoprotein template and the L protein, which has RNA-dependent RNA polymerase (RdRp), GDP polyribonucleotidyltransferase and cap-specific methyltransferase activities 2 , 3 . How P interacts with L and mediates the association with the free form of N and with the ribonucleoprotein is not clear for HMPV or other major human pathogens, including the viruses that cause measles, Ebola and rabies. Here we report a cryo-electron microscopy reconstruction that shows the ring-shaped structure of the polymerase and capping domains of HMPV-L bound to a tetramer of P. The connector and methyltransferase domains of L are mobile with respect to the core. The putative priming loop that is important for the initiation of RNA synthesis is fully retracted, which leaves space in the active-site cavity for RNA elongation. P interacts extensively with the N-terminal region of L, burying more than 4,016 Å 2 of the molecular surface area in the interface. Two of the four helices that form the coiled-coil tetramerization domain of P, and long C-terminal extensions projecting from these two helices, wrap around the L protein in a manner similar to tentacles. The structural versatility of the four P protomers—which are largely disordered in their free state—demonstrates an example of a ‘folding-upon-partner-binding’ mechanism for carrying out P adaptor functions. The structure shows that P has the potential to modulate multiple functions of L and these results should accelerate the design of specific antiviral drugs. A cryo-electron microscopy reconstruction shows the ring-shaped structure of the polymerase and capping domains of the L protein bound to a tetramer of phosphoprotein P of the human metapneumovirus.

Engineering of a bifunctional enzyme that combines DNA-dependent DNA polymerase and reverse transcriptase (RT) activities is a highly promising biotechnological goal, as it would enable one-enzyme RT-PCR. For this purpose, we selected the high-fidelity Pyrococcus furiosus (Pfu) DNA polymerase as engineering scaffold. The selection of amino acid residues for replacement was carried out based on a multi-sequence alignment of diverse DNA polymerases and literature data, which allowed us to target amino acids, which presumably are triggers of the RT activity appearance. Six mutant variants of the Pfu enzyme were created and their activity was analyzed. Through enzymatic screening, we identified the Pfu-M6 variant, which exhibits dual DNA-dependent and RNA-dependent DNA polymerase activity. This thermostable enzyme retains its inherent DNA polymerase function and has acquired the ability to catalyze reverse transcription under standard PCR conditions, which allows the created mutant form to be used for efficient amplification of DNA starting from an RNA template.

Non-homologous end joining (NHEJ) is the main repair pathway for double-strand DNA breaks (DSBs) in mammals. DNA polymerases lambda (Pol λ) and mu (Pol μ), members of the Pol X family, play a key role in this process. However, their interaction within the NHEJ complexes is unclear. Here, we present cryo-EM structures of Pol λ in complex with the DNA-PK long-range synaptic complex, and Pol μ bound to Ku70/80-DNA. These structures identify interaction sites between Ku70/80 and Pol X BRCT domains. Using mutants at the proteins interface in functional assays including cell transfection with an original gap-filling reporter, we define the role of the BRCT domain in the recruitment and activity of the two Pol X members in NHEJ and in their contribution to cell survival following DSBs. Finally, we propose a unified model for the interaction of all Pol X members with Ku70/80. The molecular basis for the enrollment of X family DNA polymerases in non-homologous end joining (NHEJ) is unclear. Here the authors elucidate the structure of Pol λ within the DNA-PK long-range complex and Pol μ in association with Ku70/80 and characterize the interaction between the BRCT domains of Pol λ and μ with Ku70/80.

RNA polymerase (Pol) III transcribes essential non-coding RNAs, including the entire pool of transfer RNAs, the 5S ribosomal RNA and the U6 spliceosomal RNA, and is often deregulated in cancer cells. The initiation of gene transcription by Pol III requires the activity of the transcription factor TFIIIB to form a transcriptionally active Pol III preinitiation complex (PIC). Here we present electron microscopy reconstructions of Pol III PICs at 3.4–4.0 Å and a reconstruction of unbound apo-Pol III at 3.1 Å. TFIIIB fully encircles the DNA and restructures Pol III. In particular, binding of the TFIIIB subunit Bdp1 rearranges the Pol III-specific subunits C37 and C34, thereby promoting DNA opening. The unwound DNA directly contacts both sides of the Pol III cleft. Topologically, the Pol III PIC resembles the Pol II PIC, whereas the Pol I PIC is more divergent. The structures presented unravel the molecular mechanisms underlying the first steps of Pol III transcription and also the general conserved mechanisms of gene transcription initiation. Detailed structures of yeast RNA polymerase III and its initiation complex shed light on how the transcription of essential non-coding RNAs begins and allow comparisons with other RNA polymerases. Structures of RNA polymerase III pre-initiation complex RNA polymerase III (Pol III) catalyses the transcription of short RNAs, including transfer RNAs and the 5S ribosomal RNA, which are essential for protein synthesis during cell growth. Pol III is predominantly regulated at the level of transcription initiation, and dysregulated Pol III activity is linked to diseases including cancer. Two independent studies in this issue, by Alessandro Vannini and colleagues and Christoph Müller and colleagues, describe electron cryo-microscopy structures of the yeast Pol III preinitiation complex, comprising the full 17-subunit Pol III and the three TFIIIB subunits (TBP, Brf1 and Bdp1) bound to promoter DNA in various functional states. The structures reveal the detailed mechanisms that underlie how Pol III is recruited to its target promoters and how promoter DNA is opened to form a stable transcription bubble, and also allow a comparison with the structures of Pol I and Pol II preinitiation complexes.

Proteins are manufactured by ribosomes—macromolecular complexes of protein and RNA molecules that are assembled within major nuclear compartments called nucleoli 1 , 2 . Existing models suggest that RNA polymerases I and III (Pol I and Pol III) are the only enzymes that directly mediate the expression of the ribosomal RNA (rRNA) components of ribosomes. Here we show, however, that RNA polymerase II (Pol II) inside human nucleoli operates near genes encoding rRNAs to drive their expression. Pol II, assisted by the neurodegeneration-associated enzyme senataxin, generates a shield comprising triplex nucleic acid structures known as R-loops at intergenic spacers flanking nucleolar rRNA genes. The shield prevents Pol I from producing sense intergenic noncoding RNAs (sincRNAs) that can disrupt nucleolar organization and rRNA expression. These disruptive sincRNAs can be unleashed by Pol II inhibition, senataxin loss, Ewing sarcoma or locus-associated R-loop repression through an experimental system involving the proteins RNaseH1, eGFP and dCas9 (which we refer to as ‘red laser’). We reveal a nucleolar Pol-II-dependent mechanism that drives ribosome biogenesis, identify disease-associated disruption of nucleoli by noncoding RNAs, and establish locus-targeted R-loop modulation. Our findings revise theories of labour division between the major RNA polymerases, and identify nucleolar Pol II as a major factor in protein synthesis and nuclear organization, with potential implications for health and disease. RNA polymerase II has an unexpected function in the nucleolus, helping to drive the expression of ribosomal RNA and to protect nucleolar structure through a mechanism involving triplex R-loop structures.

To identify approaches to target DNA repair vulnerabilities in cancer, we discovered nanomolar potent, selective, low molecular weight (MW), allosteric inhibitors of the polymerase function of DNA polymerase Polθ, including ART558. ART558 inhibits the major Polθ-mediated DNA repair process, Theta-Mediated End Joining, without targeting Non-Homologous End Joining. In addition, ART558 elicits DNA damage and synthetic lethality in BRCA1 - or BRCA2 -mutant tumour cells and enhances the effects of a PARP inhibitor. Genetic perturbation screening revealed that defects in the 53BP1/Shieldin complex, which cause PARP inhibitor resistance, result in in vitro and in vivo sensitivity to small molecule Polθ polymerase inhibitors. Mechanistically, ART558 increases biomarkers of single-stranded DNA and synthetic lethality in 53BP1-defective cells whilst the inhibition of DNA nucleases that promote end-resection reversed these effects, implicating these in the synthetic lethal mechanism-of-action. Taken together, these observations describe a drug class that elicits BRCA -gene synthetic lethality and PARP inhibitor synergy, as well as targeting a biomarker-defined mechanism of PARPi-resistance. Polθ has been recently identified as a therapeutic target in cancer but specific inhibitors are currently unavailable. Here, the authors identify small molecule inhibitors of Polθ’s polymerase activity which elicit BRCA1/2 synthetic lethality, enhance the effect of PARP inhibitors and target PARP inhibitor resistance caused by 53BP1/Shieldin pathway defects.

Key Points The proofreading exonuclease domains of the replicative DNA polymerases Pol δ and Pol ε perform an essential function in ensuring accurate DNA replication by proofreading and removing mispaired bases from the newly synthesized DNA strand. Recent studies have shown that mutations in the proofreading domains of POLD1 and POLE (which encode the catalytic subunits of Pol δ and Pol ε, respectively, in humans) predispose to colonic polyposis and cancer, and that somatic POLE proofreading domain mutations occur in several tumour types, most commonly those of the endometrium and colorectum. Interestingly, somatic POLD1 proofreading domain mutations seem to be uncommon. In several cases, the pathogenicity of these germline and somatic DNA polymerase proofreading domain mutations has been supported by studies using cell-free assays and Saccharomyces cerevisiae mutants, which confirm that they reduce or abolish exonuclease activity and increase the mutation rate. Consistent with these studies, the most striking feature of tumours with somatic POLE proofreading domain mutations is their exceptional burden of base substitution mutations — 'ultramutation'. Other notable features are their characteristic mutation spectrum, with overrepresentation of C→A transversions and, in general, a strong tendency to microsatellite stability. Endometrial cancers with somatic POLE proofreading domain mutations have an excellent prognosis, which may be because their ultramutation causes an abundance of antigenic neoepitopes, which in turn stimulate a potent antitumour immune response. The prognostic and immunological consequences of somatic POLE proofreading domain mutations in other tumour types await definition. Future studies of DNA polymerase proofreading domain mutations in cancer may provide further insights into the mechanisms and consequences of a mutator phenotype in cancer, and help to improve care for patients with endometrial, colorectal and other cancers. Recent studies have shown that germline and somatic mutations in the proofreading exonuclease domains of the replicative DNA polymerases Pol δ and Pol ε are associated with several cancers. This Review summarizes what these mutations are and how they might drive tumorigenesis, and highlights their potential as novel biomarkers and therapeutic targets. Although it has long been recognized that the exonucleolytic proofreading activity intrinsic to the replicative DNA polymerases Pol δ and Pol ε is essential for faithful replication of DNA, evidence that defective DNA polymerase proofreading contributes to human malignancy has been limited. However, recent studies have shown that germline mutations in the proofreading domains of Pol δ and Pol ε predispose to cancer, and that somatic Pol ε proofreading domain mutations occur in multiple sporadic tumours, where they underlie a phenotype of 'ultramutation' and favourable prognosis. In this Review, we summarize the current understanding of the mechanisms and consequences of polymerase proofreading domain mutations in human malignancies, and highlight the potential utility of these variants as novel cancer biomarkers and therapeutic targets.

With the ongoing COVID-19 (Coronavirus Disease 2019) pandemic, caused by the novel coronavirus SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2), there is a need for sensitive, specific, and affordable diagnostic tests to identify infected individuals, not all of whom are symptomatic. The most sensitive test involves the detection of viral RNA using RT-qPCR (quantitative reverse transcription PCR), with many commercial kits now available for this purpose. However, these are expensive, and supply of such kits in sufficient numbers cannot always be guaranteed. We therefore developed a multiplex assay using well-established SARS-CoV-2 targets alongside a human cellular control ( RPP30 ) and a viral spike-in control (Phocine Herpes Virus 1 [PhHV-1]), which monitor sample quality and nucleic acid extraction efficiency, respectively. Here, we establish that this test performs as well as widely used commercial assays, but at substantially reduced cost. Furthermore, we demonstrate >1,000-fold variability in material routinely collected by combined nose and throat swabbing and establish a statistically significant correlation between the detected level of human and SARS-CoV-2 nucleic acids. The inclusion of the human control probe in our assay therefore provides a quantitative measure of sample quality that could help reduce false-negative rates. We demonstrate the feasibility of establishing a robust RT-qPCR assay at approximately 10% of the cost of equivalent commercial assays, which could benefit low-resource environments and make high-volume testing affordable.