Single-stranded Tails Research Articles

Optimized methods for mapping DNA double-strand-break ends and resection tracts and application to meiotic recombination in mouse spermatocytes.

DNA double-strand breaks (DSBs) made by SPO11 protein initiate homologous recombination during meiosis. Subsequent to DNA strand breakage, endo- and exo-nucleases process the DNA ends to resect the strands whose 5´ termini are at the DSB, generating long 3´-terminal single-stranded tails that serve as substrates for strand exchange proteins. DSB resection is essential for meiotic recombination, but a detailed understanding of its molecular mechanism is currently lacking. Genomic approaches to mapping DSBs and resection endpoints, e.g., S1-sequencing (S1-seq) and similar methods, play a critical role in studies of meiotic DSB processing. In these methods, nuclease S1 or other enzymes that specifically degrade ssDNA are used to trim resected DSBs, allowing capture and sequencing of the ends of resection tracts. Here, we present optimization of S1-seq that improves its signal:noise ratio and allows its application to analysis of spermatocyte meiosis in adult mice. Furthermore, quantitative features of meiotic resection are evaluated for reproducibility, and we suggest approaches for analysis and interpretation of S1-seq data. We also compare S1-seq to variants that use exonuclease T and/or exonuclease VII from Escherichia coli instead of nuclease S1. Detailed step-by-step protocols and suggestions for troubleshooting are provided.

MutL Activates UvrD by Interaction Between the MutL C-terminal Domain and the UvrD 2B Domain

UvrD is a helicase vital for DNA replication and quality control processes. In its monomeric state, UvrD exhibits limited helicase activity, necessitating either dimerization or assistance from an accessory protein to efficiently unwind DNA. Within the DNA mismatch repair pathway, MutL plays a pivotal role in relaying the repair signal, enabling UvrD to unwind DNA from the strand incision site up to and beyond the mismatch. Although this interdependence is well-established, the precise mechanism of activation and the specific MutL-UvrD interactions that trigger helicase activity remain elusive. To address these questions, we employed site-specific crosslinking techniques using single-cysteine variants of MutL and UvrD followed by functional assays. Our investigation unveils that the C-terminal domain of MutL not only engages with UvrD but also acts as a self-sufficient activator of UvrD helicase activity on DNA substrates with 3′-single-stranded tails. Especially when MutL is covalently attached to the 2B or 1B domain the tail length can be reduced to a minimal substrate of 5 nucleotides without affecting unwinding efficiency.

Nonhomologous tails direct heteroduplex rejection and mismatch correction during single-strand annealing in Saccharomyces cerevisiae.

Single-strand annealing (SSA) is initiated when a double strand break (DSB) occurs between two flanking repeated sequences, resulting in a deletion that leaves a single copy of the repeat. We studied budding yeast strains carrying two 200-bp URA3 sequences separated by 2.6 kb of spacer DNA (phage lambda) in which a site-specific DSB can be created by HO or Cas9 endonucleases. Repeat-mediated deletion requires removal of long 3'-ended single-stranded tails (flaps) by Rad1-Rad10 with the assistance of Msh2-Msh3, Saw1 and Slx4. A natural 3% divergence of unequally spaced heterologies between these repeats (designated F and A) causes a significant reduction in the frequency of SSA repair. This decrease is caused by heteroduplex rejection in which mismatches (MMs) in the annealed intermediate are recognized by the MutS (Msh2 and Msh6) components of the MM repair (MMR) pathway coupled to unwinding of the duplex by the Sgs1-Rmi1-Top3 helicase. MutL homologs, Mlh1-Pms1 (MutL), are not required for rejection but play their expected role in mismatch correction. Remarkably, heteroduplex rejection is very low in strains where the divergent repeats were immediately adjacent (Tailless strains) and the DSB was induced by Cas9. These results suggest that the presence of nonhomologous tails strongly stimulates heteroduplex rejection in SSA. DNA sequencing analysis of SSA products from the FA Tailed strain showed a gradient of correction favoring the sequence opposite each 3' end of the annealed strand. Mismatches located in the center of the repair intermediate were corrected by Msh2-Msh6 mediated mismatch correction, while correction of MMs at the extremity of the SSA intermediate often appears to use a different mechanism, possibly by 3' nonhomologous tail removal that includes part of the homologous sequence. In contrast, in FA Tailless strains there was a uniform repair of the MMs across the repeat. A distinctive pattern of correction was found in the absence of MSH2, in both Tailed and Tailless strains, different from the spectrum seen in a msh3Δ msh6Δ double mutant. Previous work has shown that SSA is Rad51-independent but dependent on the strand annealing activity of Rad52. However Rad52 becomes dispensable in a Tailless construct where the DSB is induced by Cas9 or in transformation of a plasmid where SSA occurs in the absence of nonhomologous tails.

Probing and perturbing riboswitch folding using a fluorescent base analogue.

Riboswitches are mRNA segments that regulate gene expression in response to ligand binding. The Class I preQ1 riboswitch consists of a stem-loop and an adenine-rich single-stranded tail ("L3"), which adopt a pseudoknot structure upon binding of the ligand preQ1 . We inserted 2-aminopurine (2-AP), a fluorescent analogue of adenine (A), into the riboswitch at six different positions within L3. Here, 2-AP functions both as a spectroscopic probe and as a "mutation" that reveals how alteration of specific A residues impacts the riboswitch. Using fluorescence and circular dichroism spectroscopy, we found that 2-AP decreases the affinity of the riboswitch for preQ1 at all labeling positions tested, although modified and unmodified variants undergo the same global conformational changes at sufficiently high preQ1 concentration. 2-AP substitution is most detrimental to ligand binding at sites proximal to the ligand-binding pocket, while distal labeling sites exhibit the largest impacts on the stability of the L3 domain in the absence of ligand. Insertion of multiple 2-AP residues does not induce significant additional disruptions. Our results show that interactions involving the A residues in L3 play a critical role in ligand recognition by the preQ1 riboswitch and that 2-AP substitution exerts complex and varied impacts on this riboswitch.

Quantifying RNA structures and interactions with a unified reduced chain representation model

RNA is a pivotal molecule that plays critical roles in various cellular processes. Quantifying RNA structures and interactions is essential to understanding RNA function and developing RNA-based therapeutics. Using a unified five-bead model and a non-redundant database, this paper investigates the structural features and interactions of five commonly occurring RNA motifs, i.e., double-stranded helices, hairpin loops, internal/bulge loops, multi-branched junctions, and single-stranded terminal tails. Analyzing detailed distributions of RNA local structural features and base-base interactions reveals a preference for helical structures in both local backbone structures and base orientations. The interactions between adjacent bases exhibit motif-specific and sequence-dependent characteristics, reflecting the distinct topological constraints imposed by different loop-helix connection modes and the varying pairing and stacking interactions among different sequences. These findings shed light on the stability of RNA helices, emphasizing their significance in providing dominant base pairing and stacking interactions for RNA structures and stability. The four non-helix motifs encompass unpaired nucleotide loops and exhibit diverse base-base interactions, contributing to the structural diversity observed in RNA. Overall, the complexity of RNA structure arises from the intricate interplay of base-base interactions.

The hydrophilic extract from a new tomato genotype (named DHO) kills cancer cell lines through the modulation of the DNA damage response induced by Campthotecin treatment.

DNA double-strand breaks are the most toxic lesions repaired through the non-homologous and joining (NHEJ) or the homologous recombination (HR), which is dependent on the generation of single-strand tails, by the DNA end resection mechanism. The resolution of the HR intermediates leads to error-free repair (Gene Conversion) or the mutagenic pathways (Single Strand Annealing and Alternative End-Joining); the regulation of processes leading to the resolution of the HR intermediates is not fully understood. Here, we used a hydrophilic extract of a new tomato genotype (named DHO) in order to modulate the Camptothecin (CPT) DNA damage response. We demonstrated increased phosphorylation of Replication Protein A 32 Serine 4/8 (RPA32 S4/8) protein in HeLa cells treated with the CPT in combination with DHO extract with respect to CPT alone. Moreover, we pointed out a change in HR intermediates resolution from Gene Conversion to Single Strand Annealing through the modified DNA repair protein RAD52 homolog (RAD52), DNA excision repair protein ERCC-1 (ERCC1) chromatin loading in response to DHO extract, and CPT co-treatment, with respect to the vehicle. Finally, we showed an increased sensitivity of HeLa cell lines to DHO extract and CPT co-treatment suggesting a possible mechanism for increasing the efficiency of cancer therapy. We described the potential role of DHO extract in the modulation of DNA repair, in response to Camptothecin treatment (CPT), favoring an increased sensitivity of HeLa cell lines to topoisomerase inhibitor therapy.

Chromosome Segregation and Cell Division Defects in Escherichia coli Recombination Mutants Exposed to Different DNA-Damaging Treatments

Homologous recombination repairs potentially lethal DNA lesions such as double-strand DNA breaks (DSBs) and single-strand DNA gaps (SSGs). In Escherichia coli, DSB repair is initiated by the RecBCD enzyme that resects double-strand DNA ends and loads RecA recombinase to the emerging single-strand (ss) DNA tails. SSG repair is mediated by the RecFOR protein complex that loads RecA onto the ssDNA segment of gaped duplex. In both repair pathways, RecA catalyses reactions of homologous DNA pairing and strand exchange, while RuvABC complex and RecG helicase process recombination intermediates. In this work, we have characterised cytological changes in various recombination mutants of E. coli after three different DNA-damaging treatments: (i) expression of I-SceI endonuclease, (ii) γ-irradiation, and (iii) UV-irradiation. All three treatments caused severe chromosome segregation defects and DNA-less cell formation in the ruvABC, recG, and ruvABC recG mutants. After I-SceI expression and γ-irradiation, this phenotype was efficiently suppressed by the recB mutation, indicating that cytological defects result mostly from incomplete DSB repair. In UV-irradiated cells, the recB mutation abolished cytological defects of recG mutants and also partially suppressed the cytological defects of ruvABC recG mutants. However, neither recB nor recO mutation alone could suppress the cytological defects of UV-irradiated ruvABC mutants. The suppression was achieved only by simultaneous inactivation of the recB and recO genes. Cell survival and microscopic analysis suggest that chromosome segregation defects in UV-irradiated ruvABC mutants largely result from defective processing of stalled replication forks. The results of this study show that chromosome morphology is a valuable marker in genetic analyses of recombinational repair in E. coli.

Structural and DNA end resection study of the bacterial NurA-HerA complex

BackgroundThe nuclease NurA and the ATPase/translocase HerA play a vital role in repair of double-strand breaks (DSB) during the homologous recombination in archaea. A NurA-HerA complex is known to mediate DSB DNA end resection, leading to formation of a free 3′ end used to search for the homologous sequence. Despite the structures of individual archaeal types of NurA and HerA having been reported, there is limited information regarding the molecular mechanisms underlying this process. Some bacteria also possess homologs of NurA and HerA; however, the bacterial type of this complex, as well as the detailed mechanisms underlying the joining of NurA-HerA in DSB DNA end resection, remains unclear.ResultsWe report for the first time the crystal structures of Deinococcus radiodurans HerA (drHerA) in the nucleotide-free and ADP-binding modes. A D. radiodurans NurA-HerA complex structure was constructed according to a low-resolution cryo-electron microscopy map. We performed site-directed mutagenesis to map the drNurA-HerA interaction sites, suggesting that their interaction is mainly mediated by ionic links, in contrast to previously characterized archaeal NurA-HerA interactions. The key residues responsible for the DNA translocation activity, DNA unwinding activity, and catalytic activities of the drNurA-HerA complex were identified. A HerA/FtsK-specific translocation-related motif (TR motif) that guarantees the processivity of double-stranded DNA (dsDNA) translocation was identified. Moreover, a mechanism for the translocation-regulated resection of the 5′ tail of broken dsDNA and the corresponding generation of a recombinogenic 3′ single-stranded DNA tail by the drNurA-HerA complex was elucidated.ConclusionsOur work provides new insights into the mechanism underlying bacterial NurA-HerA-mediated DSB DNA end resection, and the way this complex digests the 5′ tail of a DNA duplex and provides long 3′ free end for strand invasion in the bacterial homologous recombination process.

DNA polymerase θ-mediated repair of high LET radiation-induced complex DNA double-strand breaks.

DNA polymerase θ (POLQ) is a unique DNA polymerase that is able to perform microhomology-mediated end-joining as well as translesion synthesis (TLS) across an abasic (AP) site and thymine glycol (Tg). However, the biological significance of the TLS activity is currently unknown. Herein we provide evidence that the TLS activity of POLQ plays a critical role in repairing complex DNA double-strand breaks (DSBs) induced by high linear energy transfer (LET) radiation. Radiotherapy with high LET radiation such as carbon ions leads to more deleterious biological effects than corresponding doses of low LET radiation such as X-rays. High LET-induced DSBs are considered to be complex, carrying additional DNA damage such as AP site and Tg in close proximity to the DSB sites. However, it is not clearly understood how complex DSBs are processed in mammalian cells. We demonstrated that genetic disruption of POLQ results in an increase of chromatid breaks and enhanced cellular sensitivity following treatment with high LET radiation. At the biochemical level, POLQ was able to bypass an AP site and Tg during end-joining and was able to anneal two single-stranded DNA tails when DNA lesions were located outside the microhomology. This study offers evidence that POLQ is directly involved in the repair of complex DSBs.

Mechanism of Sen1 translocation

Transcription termination is a critical step for gene regulation and genome integrity among all kingdoms of life. In <i>Saccharomyces cerevisiae</i>, one of the major termination pathways is accomplished by Sen1 helicase, a homolog to human Senataxin (SETX), defection of which raises the diseases for the central nervus system of human. Although it has been proposed that Sen1 translocates along nucleic acids by consuming adenosine triphosphates (ATPs) during termination, the mechanism for this translocation activity of Sen1 has not been well understood. In this work, our aim is to investigate the mechanism of Sen1 translocation by measuring the interactions between Sen1 and different types of nucleic acids by polyacrylamide gel electrophoresis (PAGE) assay or single-molecule Fӧrster resonance energy transfer (FRET) assay. We firstly observe the unwinding activity of Sen1 on a tailed duplex DNA in the presence of 1 mM ATP via PAGE assay, where the translocation activity of Sen1 is involved. As the binding activity is crucial for translocation, then we examine the binding affinity of Sen1 to the single-stranded DNA via PAGE assay, revealing a stable binding of Sen1 with an occupied length of nucleic acids of less than 24 nt. In the presence of 1 µM ATP, we observe that Sen1 dynamically binds to and dissociates from the tailed duplex DNA in the single-molecule FRET assay. By titrating ATP concentrations from 1–500 µM, we observe a gradual decrease in the mean durations of Sen1 binding, suggesting an ATP-dependent binding affinity of Sen1 to single-stranded DNA. We then fit these mean durations to the classical Michaelis-Menten model and obtain a minimum binding duration of (0.18 ± 0.01) s at saturating ATP concentrations and <i>K</i><sub>m</sub> of (13.1 ± 0.1) µM for the ATP-dependent binding of Sen1. This result is consistent with that from a translocation activity of Sen1. Taking into account the translocation length of the half of the single-stranded tail, i.e. 13 nt, a mean rate of 70 nt/s is estimated. Reversing the translocation direction, we observe an increase in the duration of Sen1 binding to the single-stranded tail, which suggests an impediment of DNA duplex in front of Sen1 translocation or the possible duplex DNA unwinding activity of Sen1. Our quantitative measurements on Sen1 translocation are helpful in deepening our understanding of the mechanism of eukaryotic transcription termination by Sen1.

Rad51-mediated interhomolog recombination during budding yeast meiosis is promoted by the meiotic recombination checkpoint and the conserved Pif1 helicase.

During meiosis, recombination between homologous chromosomes (homologs) generates crossovers that promote proper segregation at the first meiotic division. Recombination is initiated by Spo11-catalyzed DNA double strand breaks (DSBs). 5' end resection of the DSBs creates 3' single strand tails that two recombinases, Rad51 and Dmc1, bind to form presynaptic filaments that search for homology, mediate strand invasion and generate displacement loops (D-loops). D-loop processing then forms crossover and non-crossover recombinants. Meiotic recombination occurs in two temporally distinct phases. During Phase 1, Rad51 is inhibited and Dmc1 mediates the interhomolog recombination that promotes homolog synapsis. In Phase 2, Rad51 becomes active and functions with Rad54 to repair residual DSBs, making increasing use of sister chromatids. The transition from Phase 1 to Phase 2 is controlled by the meiotic recombination checkpoint through the meiosis-specific effector kinase Mek1. This work shows that constitutive activation of Rad51 in Phase 1 results in a subset of DSBs being repaired by a Rad51-mediated interhomolog recombination pathway that is distinct from that of Dmc1. Strand invasion intermediates generated by Rad51 require more time to be processed into recombinants, resulting in a meiotic recombination checkpoint delay in prophase I. Without the checkpoint, Rad51-generated intermediates are more likely to involve a sister chromatid, thereby increasing Meiosis I chromosome nondisjunction. This Rad51 interhomolog recombination pathway is specifically promoted by the conserved 5'-3' helicase PIF1 and its paralog, RRM3 and requires Pif1 helicase activity and its interaction with PCNA. This work demonstrates that (1) inhibition of Rad51 during Phase 1 is important to prevent competition with Dmc1 for DSB repair, (2) Rad51-mediated meiotic recombination intermediates are initially processed differently than those made by Dmc1, and (3) the meiotic recombination checkpoint provides time during prophase 1 for processing of Rad51-generated recombination intermediates.

Significant destabilization of human telomeric G-quadruplex upon peptide binding: dramatic effect of flanking bases

Human telomere is composed of highly repeated hexanucleotide sequence TTAGGG and a 3′ single-stranded DNA tail. Many telomere G4 topologies characterized at atomic level by X-ray crystallography and NMR studies. Until now, various small ligands developed to interact with G-quadruplex mainly to stabilize the structure and least is known for its destabilization. In this study, we provide the first evidence of human telomeric G4 destabilization upon peptide binding in dilute and cell-mimicking molecular crowing conditions due to the changes in flanking bases of human telomeric sequences. Hence, our findings will open the new ways to target diseases related with increasing the efficiency of DNA replication, transcription or duplex reannealing. Communicated by Ramaswamy H. Sarma

Fast DNA biosensing based on isothermal amplification, unmodified gold nanoparticles, and smartphone detection

The protection of the consumer against foodborne illnesses and fraudulent practices requires methods capable of detecting critical components. Classical instrumental and DNA-based technologies have assay time, portability, and cost limitations. Thus, food control needs alternative methodologies for massive and cost-effective screening. Herein, we report an instrument-free method based on detecting genes that encode proteins related to food allergies and ingredients associated with illegal practices. Tailed recombinase polymerase amplification (tailed-RPA) provided the selective isothermal amplification of target regions. A reproducible and fast optical detection was developed based on the salt-induced aggregation of 15 nm gold nanoparticles (AuNPs). The target presence was directly observed (naked-eye detection) and quantified using a smartphone and RGB image decomposition. The advantages arise from the effective formation of a hybrid complex between non-functionalized nanoparticles and amplification products with a single-strand tail, featuring short incubation time, simplicity, and low sample and reagent volumes. As proof of concept, two targets were determined: trnL gene and ITS region. The first sequence is located in the chloroplast genome of cereals and is valuable to control the adulteration of meat products. The second is a fragment common to the three main gluten-containing cereals, applicable to the indirect detection of this allergen. The novelty also is the integration of a low-cost assay platform and a straightforward interpretation system to foster its implementation in the industry. The RPA-AuNP assay offered a good sensitivity (genomic DNA 0.8 ng), selectivity (absence of unspecific response), reproducibility (standard deviation 2–11%), and accuracy in marketed food products (100%). Therefore, a competitive biosensing system enables better control according to food industry/consumers demands from Farm to Fork strategy.

Abstract P109: Targeting BRD4 in T cells with self-delivering RNAi PH-894 for immunotherapy

Abstract BRD4 is a member of the family of BET proteins that act as epigenetic readers and regulators of gene transcription. BRD4 is a known driver of tumor growth via enhancement of transcription of oncogenes such as Myc. More recent evidence also suggests a role of BRD4 in T cells. T cell differentiation and T cell exhaustion is epigenetically regulated and BRD4 has been associated with T cell differentiation. Available small molecule inhibitors of BET proteins do not target BRD4 specifically, but also inhibit other members of the BET protein family. We therefore investigated the effects of BRD4 silencing on T cells with the INTASYL™ compound PH-894, a self-delivering RNAi specifically targeting BRD4. INTASYL self-delivering RNAi molecules consist of an asymmetric duplex structure, a small duplex region (≤ 15 base pairs), a single-stranded phosphorothioate tail, and chemical modifications to confer stability, hydrophobicity, and efficient cellular uptake. Primary human T cells from healthy donors, either unstimulated or activated by OKT3 or anti-CD3/CD28 beads, were treated with PH-894. Additional studies were performed with CD8+ T cells magnetically sorted from primary human T cells. Silencing of BRD4 mRNA was assessed by RT-qPCR, and protein by flow cytometry. Phenotypic markers of T cell activation, differentiation and function were assessed by flow cytometry and cytokine secretion by cytometric bead array. Expanded and PH-894-treated CD8+ T cells were studied for IFNγ production upon coculture with allogeneic melanoma cell lines. Silencing of BRD4 mRNA and protein by PH-894 was concentration dependent and durable for up to 10 days in activated and naïve T cells. Silencing was specific for BRD4 with no effect on BRD2 or BRD3 expression. In activated T cells there was a suppression of IL-10 secretion and no effect on inflammatory cytokine levels. Markers of T cell activation were elevated on naïve PH-894-treated T cells with a shift to an increased CD8+/CD4+ ratio. In addition, BRD4 silencing in activated CD8+ T cells coincided with a decreased expression of transcription factors typically induced immediately upon TCR activation and are known drivers of T cell differentiation towards an effector phenotype. Moreover, CD8+ T cells expanded in the presence of PH-894 produced increased levels of IFNγ when cocultured with two allogeneic melanoma cell lines. These data translated in vivo with PH-894 treatment eliciting a dose associated inhibition of tumor growth in a CT26 murine colon carcinoma model. Ex vivo analysis of tumors found PH-894 efficacy was associated with decreased expression of BRD4 in tumor infiltrating CD3 T cells, and a reduction of Foxp3hi CD25hi CD4+ CD3+ Tregs, compared with control tumors. Specific silencing of BRD4 with PH-894 resulted in phenotypic changes on T cells associated with T cell activation and reduced immunosuppression. These data suggest that BRD4 not only plays a role on tumor cells but can also regulate T cell function and that PH-894 can reprogram T cells to provide enhanced immunotherapeutic activity. Citation Format: Melissa Maxwell, Daisuke Ujiie, Benjamin Cuiffo, Brianna Rivest, Dingxue Yan, James Cardia, Jeroen Melief, Rolf Kiessling, Simon P. Fricker. Targeting BRD4 in T cells with self-delivering RNAi PH-894 for immunotherapy [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P109.

Multiple hPOT1-TPP1 cooperatively unfold contiguous telomeric G-quadruplexes proceeding from 3' toward 5', a feature due to a 3'-end binding preference and to structuring of telomeric DNA.

Telomeres are DNA repeated sequences that associate with shelterin proteins and protect the ends of eukaryotic chromosomes. Human telomeres are composed of 5′TTAGGG repeats and ends with a 3′ single-stranded tail, called G-overhang, that can be specifically bound by the shelterin protein hPOT1 (human Protection of Telomeres 1). In vitro studies have shown that the telomeric G-strand can fold into stable contiguous G-quadruplexes (G4). In the present study we investigated how hPOT1, in complex with its shelterin partner TPP1, binds to telomeric sequences structured into contiguous G4 in potassium solutions. We observed that binding of multiple hPOT1–TPP1 preferentially proceeds from 3′ toward 5′. We explain this directionality in terms of two factors: (i) the preference of hPOT1–TPP1 for the binding site situated at the 3′ end of a telomeric sequence and (ii) the cooperative binding displayed by hPOT1–TPP1 in potassium. By comparing binding in K+ and in Li+, we demonstrate that this cooperative behaviour does not stem from protein-protein interactions, but from structuring of the telomeric DNA substrate into contiguous G4 in potassium. Our study suggests that POT1-TPP1, in physiological conditions, might preferentially cover the telomeric G-overhang starting from the 3′-end and proceeding toward 5′.

Polymerase θ Coordinates Multiple Intrinsic Enzymatic Activities during DNA Repair.

The POLQ gene encodes DNA polymerase θ, a 2590 amino acid protein product harboring DNA-dependent ATPase, template-dependent DNA polymerase, dNTP-dependent endonuclease, and 5′–dRP lyase functions. Polymerase θ participates at an essential step of a DNA double-strand break repair pathway able to join 5′-resected substrates by locating and pairing microhomologies present in 3′-overhanging single-stranded tails, cleaving the extraneous 3′-DNA by dNTP-dependent end-processing, before extending the nascent 3′ end from the microhomology annealing site. Metazoans require polymerase θ for full resistance to DNA double-strand break inducing agents but can survive knockout of the POLQ gene. Cancer cells with compromised homologous recombination, or other DNA repair defects, over-utilize end-joining by polymerase θ and often over-express the POLQ gene. This dependency points to polymerase θ as an ideal drug target candidate and multiple drug-development programs are now preparing to enter clinical trials with small-molecule inhibitors. Specific inhibitors of polymerase θ would not only be predicted to treat BRCA-mutant cancers, but could thwart accumulated resistance to current standard-of-care cancer therapies and overcome PARP-inhibitor resistance in patients. This article will discuss synthetic lethal strategies targeting polymerase θ in DNA damage-response-deficient cancers and summarize data, describing molecular structures and enzymatic functions.

U7 deciphered: the mechanism that forms the unusual 3′ end of metazoan replication-dependent histone mRNAs

In animal cells, replication-dependent histone mRNAs end with a highly conserved stem-loop structure followed by a 4- to 5-nucleotide single-stranded tail. This unique 3' end distinguishes replication-dependent histone mRNAs from all other eukaryotic mRNAs, which end with a poly(A) tail produced by the canonical 3'-end processing mechanism of cleavage and polyadenylation. The pioneering studies of Max Birnstiel's group demonstrated nearly 40 years ago that the unique 3' end of animal replication-dependent histone mRNAs is generated by a distinct processing mechanism, whereby histone mRNA precursors are cleaved downstream of the stem-loop, but this cleavage is not followed by polyadenylation. The key role is played by the U7 snRNP, a complex of a ∼60 nucleotide U7 snRNA and many proteins. Some of these proteins, including the enzymatic component CPSF73, are shared with the canonical cleavage and polyadenylation machinery, justifying the view that the two metazoan pre-mRNA 3'-end processing mechanisms have a common evolutionary origin. The studies on U7 snRNP culminated in the recent breakthrough of reconstituting an entirely recombinant human machinery that is capable of accurately cleaving histone pre-mRNAs, and determining its structure in complex with a pre-mRNA substrate (with 13 proteins and two RNAs) that is poised for the cleavage reaction. The structure uncovered an unanticipated network of interactions within the U7 snRNP and a remarkable mechanism of activating catalytically dormant CPSF73 for the cleavage. This work provides a conceptual framework for understanding other eukaryotic 3'-end processing machineries.

A strip of lateral flow gene assay using gold nanoparticles for point-of-care diagnosis of African swine fever virus in limited environment.

Recombinase polymerase amplification (RPA) was combined with lateral flow to develop a gold nanoparticles test strip for point-of-care diagnosis of African swine fever virus (ASFV), which is called lateral flow gene assay (LFGA). Common diagnostic techniques, including polymerase chain reaction (PCR) and immunochromatography, are time-consuming and labor-intensive, and generally require costly instruments. For improvement, this assay used tailed primers to produce DNA duplexes with a single-stranded tail at one end which can hybridize with a gold nanoparticle (AuNP)-labeled oligonucleotide detection probe. And then, biotin attached to the other end of the product bound to streptavidin, which previously fixed to the test line. Therefore, there would form a sandwich structure, and gold nanoparticles labeled on the detection probe would show a red band on the test line of strip. With the low reaction temperature (37~42°C) and short reaction time (30min), LFGA can specifically identify ASFV in blood samples infected with ASFV and classical swine fever virus (CSFV), and the LOD was 102 copies/μL, which was comparable to that of agarose gel electrophoresis. In addition, blood samples infected with ASFV and CSFV were tested, and it was found that the LFGA can specifically identify ASFV DNA. In conclusion, LFGA achieves visual observation of the product after rapid RPA amplification and does not require any expensive instruments during the entire process, which is very helpful for early diagnosis of ASFV. Combined recombinase polymerase amplification (RPA) with lateral flow, we developed a gold nanoparticles test strip for point-of-care diagnosis of African swine fever virus. The upstream primers of RPA were modified with biotin, and the downstream primers were modified with a C3 spacer and an oligonucleotide tail that can be hybridized to a gold nanoparticle-labeled oligonucleotide detection probe. On the strip, the test line and control line were sprayed with streptavidin and an oligonucleotide control probe. In the presence of positive products, RPA products can form a sandwich structure on the test line. Therefore, two red lines will be displayed both on the test line and control line. When there is no positive product, only the control line is shown in red. Its low reaction temperature (37~42°C) and short time of amplification and detection (30min) make ASFV realizing point-of-care diagnosis in limited environment.

Ferrocene-Containing DNA Monolayers: Influence of Electrostatics on the Electron Transfer Dynamics.

A 153-mer target DNA was amplified using ethynyl ferrocene dATP and a tailed forward primer resulting in a duplex with a single-stranded DNA tail for hybridization to a surface-tethered probe. A thiolated probe containing the sequence complementary to the tail as well as a 15 polythimine vertical spacer with a (CH2)6 spacer was immobilized on the surface of a gold electrode and hybridized to the ferrocene-modified complementary strand. Potential step chronoamperometry and cyclic voltammetry were used to probe the potential of zero charge, PZC, and the rate of heterogeneous electron transfer between the electrode and the immobilized ferrocene moieties. Chronoamperometry gives three, well-resolved exponential current–time decays corresponding to ferrocene centers located within 13 Å (4 bases) along the duplex. Significantly, the apparent standard heterogeneous electron transfer rate constant, kappo, observed depends on the initial potential, i.e., the rate of electron transfer at zero driving force is not the same for oxidation and reduction of the ferrocene labels. Moreover, the presence of ions, such as Sr2+, that strongly ion pair with the negatively charged DNA backbone modulates the electron transfer rate significantly. Specifically, kappo = 246 ± 23.5 and 14 ± 1.2 s–1 for reduction and oxidation, respectively, where the Sr2+ concentration is 10 mM, but the corresponding values in 1 M Sr2+ are 8 ± 0.8 and 150 ± 12 s–1. While other factors may be involved, these results are consistent with a model in which a low Sr2+ concentration and an initial potential that is negative of the PZC lead to electrostatic repulsion of the negatively charged DNA backbone and the negatively charged electrode. This leads to the DNA adopting an extended configuration (concertina open), resulting in a slow rate of heterogeneous electron transfer. In contrast, for ferrocene reduction, the initial potential is positive of PZC and the negatively charged DNA is electrostatically attracted to the electrode (concertina closed), giving a shorter electron transfer distance and a higher rate of heterogeneous electron transfer. When the Sr2+ concentration is high, the charge on the DNA backbone is compensated by the electrolyte and the charge on the electrode dominates the electron transfer dynamics and the opposite potential dependence is observed. These results open up the possibility of electromechanical switching using DNA superstructures.

Combination of ferrocene decorated gold nanoparticles and engineered primers for the direct reagentless determination of isothermally amplified DNA.

A reagent-less DNA sensor has been developedexploiting a combination of gold nanoparticles, modified primers, and isothermal amplification. It is applied to the determination ofKarlodinium armiger, a toxic microalgae, as a model analyte to demonstrate this generic platform. Colloidal gold nanoparticles with an average diameter of 14 ± 0.87nm were modified with a mixed self-assembled monolayer of thiolated 33-mer DNA probes and (6-mercaptohexyl) ferrocene. Modified primers, exploiting a C3 spacer between the primer-binding site and an engineered single-stranded tail, were used in an isothermal recombinase polymerase amplification reaction to produce an amplicon by two single-stranded tails. These tails were designed to be complementary to a gold electrode tethered capture oligo probe, and an oligo probe immobilized on the gold nanoparticles, respectively. The time required for hybridization of the target tailed DNA with the surface immobilized probe and reporter probe immobilized on AuNPs was optimized and reduced to 10min, in both cases. Amplification time was further optimized to be 40min to ensure the maximum signal. Under optimal conditions, the limit of detection was found to be 1.6 fM of target dsDNA. Finally, the developed biosensor was successfully applied to the detection of genomic DNA extracted from a seawater sample that had been spiked with K. armiger cells. The demonstrated generic electrochemical genosensor can be exploited for the detection of any DNA sequence and ongoing work is moving towards an integrated system for use at the point-of-need.