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Nanopore-based DNA analysis is a new single-molecule technique that involves monitoring the flow of ions through a narrow pore, and detecting changes in this flow as DNA molecules also pass through the pore. It has the potential to carry out a range of laboratory and medical DNA analyses, orders of magnitude faster than current methods. Initial experiments used a protein channel for its pre-defined, precise structure, but since then several approaches for the fabrication of solid-state pores have been developed. These aim to match the capabilities of biochannels, while also providing increased durability, control over pore geometry and compatibility with semiconductor and microfluidics fabrication techniques. This review summarizes each solid-state nanopore fabrication technique reported to date, and compares their advantages and disadvantages. Methods and applications for nanopore surface modification are also presented, followed by a discussion of approaches used to measure pore size, geometry and surface properties. The review concludes with an outlook on the future of solid-state nanopores.

Nanopore-based DNA analysis is a single-molecule technique with revolutionary potential. It promises to carry out a range of analyses, orders of magnitude faster than current methods, including length measurement, specific sequence detection, single-molecule dynamics and even sequencing. The concept involves using an applied voltage to drive DNA molecules through a narrow pore that separates chambers of electrolyte solution. This voltage also drives a flow of electrolyte ions through the pore, measured as an electric current. When molecules pass through the pore, they block the flow of ions and, thus, their structure and length can be determined based on the degree and duration of the resulting current reductions. In this review, I explain the nanopore-based DNA analysis concept and briefly explore its historical foundations, before discussing and summarizing all experimental results reported to date. I conclude with a summary of the obstacles that must be overcome for it to realize its promised potential.

Ataxia with oculomotor apraxia 2 (AOA-2) and amyotrophic lateral sclerosis (ALS4) are neurological disorders caused by mutations in the gene encoding for senataxin (SETX), a putative RNA:DNA helicase involved in transcription and in the maintenance of genome integrity. Here, using ChIP followed by high throughput sequencing (ChIP-seq), we report that senataxin is recruited at DNA double-strand breaks (DSBs) when they occur in transcriptionally active loci. Genome-wide mapping unveiled that RNA:DNA hybrids accumulate on DSB-flanking chromatin but display a narrow, DSB-induced, depletion near DNA ends coinciding with senataxin binding. Although neither required for resection nor for timely repair of DSBs, senataxin was found to promote Rad51 recruitment, to minimize illegitimate rejoining of distant DNA ends and to sustain cell viability following DSB production in active genes. Our data suggest that senataxin functions at DSBs in order to limit translocations and ensure cell viability, providing new insights on AOA2/ALS4 neuropathies. Recent studies suggest key roles of RNA in DNA double-strand breaks repair. Here the authors identify the helicase senataxin to be involved in DNA repair and resolve RNA:DNA hybrids forming at DNA double-strand breaks.

The contribution of lncRNAs to tumour progression and the regulatory mechanisms driving their expression are areas of intense investigation. Here, we characterize the binding of heterogeneous nuclear ribonucleoprotein E1 (hnRNP E1) to a nucleic acid structural element located in exon 12 of PNUTS (also known as PPP1R10) pre-RNA that regulates its alternative splicing. HnRNP E1 release from this structural element, following its silencing, nucleocytoplasmic translocation or in response to TGFβ, allows alternative splicing and generates a non-coding isoform of PNUTS . Functionally the lncRNA- PNUTS serves as a competitive sponge for miR-205 during epithelial–mesenchymal transition (EMT). In mesenchymal breast tumour cells and in breast tumour samples, the expression of lncRNA- PNUTS is elevated and correlates with levels of ZEB mRNAs. Thus, PNUTS is a bifunctional RNA encoding both PNUTS mRNA and lncRNA- PNUTS , each eliciting distinct biological functions. While PNUTS mRNA is ubiquitously expressed, lncRNA- PNUTS appears to be tightly regulated dependent on the status of hnRNP E1 and tumour context. Grelet et al. find that hnRNP E1 release from PNUTS pre-RNA in response to TGFβ generates a lncRNA that acts as competitive sponge for miR-205, promoting epithelial–mesenchymal transition in cancer.

The 11-subunit RNA exosome is thought to regulate the mammalian noncoding transcriptome; here, a mouse model is generated in which the essential Exosc3 subunit of the RNA exosome in B cells is conditionally deleted, revealing a link between sites of genomic RNA exosome function and AID-mediated chromosomal translocations. Noncoding RNAs pinpoint AID in B cells It is difficult to identify rare non-coding RNA (ncRNA) species because of their low abundance in cells and the fact that they are rapidly degraded, mainly through the action of the cellular non-coding RNA 3′–5′ degradation complex, RNA exosome. Uttiya Basu and colleagues have generated a mouse model in which an essential subunit (exosome component 3, Exosc3 ) of the RNA exosome can be conditionally inactivated in B cells. Exosc3-deficient B cells lack the recombination and mutagenesis activities that are necessary for generating antibodies. Many non-coding RNAs normally degraded are found in these cells, including xTSS-RNAs, a type of antisense RNA encoded at transcription start sites. Surprisingly, the locations of the xTSS-RNAs correlate with sites of translocation breakages. The model suggested is that antisense transcription of the ncRNAs recruits activation-induced cytidine deaminase (AID) and results in formation of single-strand DNA; pairing with the RNAs makes R-loops that can lead to genomic instability. The vast majority of the mammalian genome has the potential to express noncoding RNA (ncRNA). The 11-subunit RNA exosome complex is the main source of cellular 3′–5′ exoribonucleolytic activity and potentially regulates the mammalian noncoding transcriptome 1 . Here we generated a mouse model in which the essential subunit Exosc3 of the RNA exosome complex can be conditionally deleted. Exosc3 -deficient B cells lack the ability to undergo normal levels of class switch recombination and somatic hypermutation, two mutagenic DNA processes used to generate antibody diversity via the B-cell mutator protein activation-induced cytidine deaminase (AID) 2 , 3 . The transcriptome of Exosc3 -deficient B cells has revealed the presence of many novel RNA exosome substrate ncRNAs. RNA exosome substrate RNAs include xTSS-RNAs, transcription start site (TSS)-associated antisense transcripts that can exceed 500 base pairs in length and are transcribed divergently from cognate coding gene transcripts. xTSS-RNAs are most strongly expressed at genes that accumulate AID-mediated somatic mutations and/or are frequent translocation partners of DNA double-strand breaks generated at Igh in B cells 4 , 5 . Strikingly, translocations near TSSs or within gene bodies occur over regions of RNA exosome substrate ncRNA expression. These RNA exosome-regulated, antisense-transcribed regions of the B-cell genome recruit AID and accumulate single-strand DNA structures containing RNA–DNA hybrids. We propose that RNA exosome regulation of ncRNA recruits AID to single-strand DNA-forming sites of antisense and divergent transcription in the B-cell genome, thereby creating a link between ncRNA transcription and overall maintenance of B-cell genomic integrity.

It is not clear how spontaneous DNA double-strand breaks (DSBs) form and are processed in normal cells, and whether they predispose to cancer-associated translocations. We show that DSBs in normal mammary cells form upon release of paused RNA polymerase II (Pol II) at promoters, 5′ splice sites and active enhancers, and are processed by end-joining in the absence of a canonical DNA-damage response. Logistic and causal-association models showed that Pol II pausing at long genes is the main predictor and determinant of DSBs. Damaged introns with paused Pol II-pS5, TOP2B and XRCC4 are enriched in translocation breakpoints, and map at topologically associating domain boundary-flanking regions showing high interaction frequencies with distal loci. Thus, in unperturbed growth conditions, release of paused Pol II at specific loci and chromatin territories favors DSB formation, leading to chromosomal translocations. Release of paused Pol II at specific intronic loci or chromatin domains favors the formation of abnormal DNA recombination, leading to cancer-associated chromosomal translocations.

Remdesivir is the only FDA-approved drug for the treatment of COVID-19 patients. The active form of remdesivir acts as a nucleoside analog and inhibits the RNA-dependent RNA polymerase (RdRp) of coronaviruses including SARS-CoV-2. Remdesivir is incorporated by the RdRp into the growing RNA product and allows for addition of three more nucleotides before RNA synthesis stalls. Here we use synthetic RNA chemistry, biochemistry and cryo-electron microscopy to establish the molecular mechanism of remdesivir-induced RdRp stalling. We show that addition of the fourth nucleotide following remdesivir incorporation into the RNA product is impaired by a barrier to further RNA translocation. This translocation barrier causes retention of the RNA 3ʹ-nucleotide in the substrate-binding site of the RdRp and interferes with entry of the next nucleoside triphosphate, thereby stalling RdRp. In the structure of the remdesivir-stalled state, the 3ʹ-nucleotide of the RNA product is matched and located with the template base in the active center, and this may impair proofreading by the viral 3ʹ-exonuclease. These mechanistic insights should facilitate the quest for improved antivirals that target coronavirus replication. Remdesivir is a nucleoside analog that inhibits the SARS-CoV-2 RNA dependent RNA polymerase (RdRp) and is used as a drug to treat COVID19 patients. Here, the authors provide insights into the mechanism of remdesivir-induced RdRp stalling by determining the cryo-EM structures of SARS-CoV-2 RdRp with bound RNA molecules that contain remdesivir at defined positions and observe that addition of the fourth nucleotide following remdesivir incorporation into the RNA product is impaired by a barrier to further RNA translocation.

The journey of RNA polymerase II (Pol II) as it transcribes a gene is anything but a smooth ride. Transcript elongation is discontinuous and can be perturbed by intrinsic regulatory barriers, such as promoter-proximal pausing, nucleosomes, RNA secondary structures and the underlying DNA sequence. More substantial blocking of Pol II translocation can be caused by other physiological circumstances and extrinsic obstacles, including other transcribing polymerases, the replication machinery and several types of DNA damage, such as bulky lesions and DNA double-strand breaks. Although numerous different obstacles cause Pol II stalling or arrest, the cell somehow distinguishes between them and invokes different mechanisms to resolve each roadblock. Resolution of Pol II blocking can be as straightforward as temporary backtracking and transcription elongation factor S-II (TFIIS)-dependent RNA cleavage, or as drastic as premature transcription termination or degradation of polyubiquitylated Pol II and its associated nascent RNA. In this Review, we discuss the current knowledge of how these different Pol II stalling contexts are distinguished by the cell, how they overlap with each other, how they are resolved and how, when unresolved, they can cause genome instability.Transcript elongation by RNA polymerase II can be perturbed by barriers such as promoter-proximal pausing and nucleosomes and by obstacles such as the replication machinery and DNA lesions. Recent studies revealed how different contexts of RNA polymerase II stalling are distinguished and resolved, and how unresolved stalling can cause genome instability.

Rho is a ring-shaped hexameric ATP-dependent molecular motor. Together with the transcription elongation factor NusG, Rho mediates factor-dependent transcription termination and transcription–translation-coupling quality control in Escherichia coli 1 – 4 . Here we report the preparation of complexes that are functional in factor-dependent transcription termination from Rho, NusG, RNA polymerase (RNAP), and synthetic nucleic acid scaffolds, and we report cryogenic electron microscopy structures of the complexes. The structures show that functional factor-dependent pre-termination complexes contain a closed-ring Rho hexamer; have RNA threaded through the central channel of Rho; have 60 nucleotides of RNA interacting sequence-specifically with the exterior of Rho and 6 nucleotides of RNA interacting sequence-specifically with the central channel of Rho; have Rho oriented relative to RNAP such that ATP-dependent translocation by Rho exerts mechanical force on RNAP; and have NusG bridging Rho and RNAP. The results explain five decades of research on Rho and provide a foundation for understanding Rho’s function. Structures presented in this study confirm decades of genetic and biochemical evidence for the mechanism of Rho-dependent termination in bacteria.

Cryo-electron microscopy analysis of yeast Rad26 bound to RNA polymerase II provides insight into the initiation of the transcription-coupled DNA repair mechanism in eukaryotes. Transcription-coupled repair complex Transcription-coupled DNA repair removes DNA lesions from the template strand that present obstacles to the translocation of RNA polymerase II (Pol II). The process is initiated by the recruitment of the Cockayne syndrome group B (CSB) protein in humans—or the equivalent Rad26 in the yeast ( Saccharomyces cerevisiae )—to the arrested polymerase complex. Here, Andres Leschziner, Dong Wang and colleagues have used cryo-electron microscopy to solve the structure of a complex of S. cerevisiae Rad26 bound to Pol II. Rad26 binds to DNA upstream of Pol II and causes marked bending of the DNA, and the Swi2/Snf2-family ATPase domain of Rad26 is proposed to promote forward movement of Pol II. The authors suggest a mechanistic model whereby Rad26 ensures transcription-coupled recognition of DNA lesions while also functioning as a transcription elongation factor. Eukaryotic transcription-coupled repair (TCR) is an important and well-conserved sub-pathway of nucleotide excision repair that preferentially removes DNA lesions from the template strand that block translocation of RNA polymerase II (Pol II) 1 , 2 . Cockayne syndrome group B (CSB, also known as ERCC6) protein in humans (or its yeast orthologues, Rad26 in Saccharomyces cerevisiae and Rhp26 in Schizosaccharomyces pombe ) is among the first proteins to be recruited to the lesion-arrested Pol II during the initiation of eukaryotic TCR 1 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 . Mutations in CSB are associated with the autosomal-recessive neurological disorder Cockayne syndrome, which is characterized by progeriod features, growth failure and photosensitivity 1 . The molecular mechanism of eukaryotic TCR initiation remains unclear, with several long-standing unanswered questions. How cells distinguish DNA lesion-arrested Pol II from other forms of arrested Pol II, the role of CSB in TCR initiation, and how CSB interacts with the arrested Pol II complex are all unknown. The lack of structures of CSB or the Pol II–CSB complex has hindered our ability to address these questions. Here we report the structure of the S. cerevisiae Pol II–Rad26 complex solved by cryo-electron microscopy. The structure reveals that Rad26 binds to the DNA upstream of Pol II, where it markedly alters its path. Our structural and functional data suggest that the conserved Swi2/Snf2-family core ATPase domain promotes the forward movement of Pol II, and elucidate key roles for Rad26 in both TCR and transcription elongation.