Double-stranded Research Articles

Therapeutic siRNA Loaded to RISC as Single and Double Strands Requires an Appropriate Quantitative Assay for RISC PK Assessment.

In recent years, therapeutic siRNA projects are booming in the biotech and pharmaceutical industries. As these drugs act by silencing the target gene expression, a critical step is the binding of antisense strands of siRNA to RNA-induced silencing complex (RISC) and then degrading their target mRNA. However, data that we recently obtained suggest that double-stranded siRNA can also load to RISC. This brings a new understanding of the mechanism of RISC loading which may have a potential impact on how quantification of RISC loaded siRNA should be performed. By combining RNA immune precipitation and probe-based hybridization LC-fluorescence approach, we have developed a novel assay that can accurately quantify the RISC-bound antisense strand, irrespective of which form (double-stranded or single-stranded) is loaded on RISC. In addition, this novel assay can discriminate between the 5'-phosphorylated antisense (5'p-AS) and the nonphosphorylated forms, therefore specifically quantifying the RISC bound 5'p-AS. In comparison, stem-loop qPCR assay does not provide discrimination and accurate quantification when the oligonucleotide analyte exists as a mixture of double and single-stranded forms. Taking together, RISC loading assay with probe-hybridization LC-fluorescence technique would be a more accurate and specific quantitative approach for RISC-associated pharmacokinetic assessment.

The RAD51 S181P mutation shortens lifespan of female mice

RAD51 is critical to the homologous recombination (HR) pathway that repairs DNA double strand breaks (DSBs) and protects replication forks (RFs). Previously, we showed that the S181P (SP) mutation in RAD51 causes defective RF maintenance but is proficient for DSB repair. Here we report that SP/SP female mice exhibit a shortened lifespan compared to +/+ females but not males. Histological analysis found that most mice in this study died from lymphoma, independent of genotype and sex. We propose that a potential cause for shortened lifespan in SP/SP females is due to the RF defect.

Superior Capsular Reconstruction using the Long Head of Biceps Tendon: A Biomechanical Assessment of Tenodesis Location and Angle of Fixation

BackgroundMassive irreparable rotator cuff tears (MIRCT) treated with superior capsular reconstruction (SCR) using the long head of the biceps tendon have shown satisfactory early results. Different techniques and positions for biceps tenodesis have been described. This study aimed to evaluate the effect of tenodesis location and glenohumeral fixation angle for graft tensioning on the biomechanics of a SCR using a single strand biceps technique. MethodsEight cadaveric shoulders were mounted to a custom biomechanical simulator which employed static tone loads to the deltoid and rotator cuff muscles. All cadavers were first tested in the intact condition, and then in the simulated MIRCT condition by sectioning the tendinous insertions of the supraspinatus and upper border of the infraspinatus. SCR using the long head of the biceps tendon was then evaluated. Three biceps tenodesis locations relative to the greater tuberosity (anterior, middle, and posterior) and two glenohumeral fixation angles (0° and 30°) for graft tensioning were tested. An optical tracking system was used to quantify superior-inferior (SI) and anterior-posterior (AP) humeral head translation relative to the glenoid, while the functional abduction force was quantified using a load sensor. All tests were conducted at 0°, 30° and 60° of glenohumeral abduction in a randomized fashion. ResultsWhen assessing isolated superior humeral head migration, all biceps tenodesis locations were effective at decreasing superior migration, with no tenodesis location significantly better than the other (P=0.213). However, biceps grafts tensioned at 30° of glenohumeral abduction were significantly better at reducing proximal humeral migration as compared to graft tensioning at 0° abduction (P=0.008).Posterior humeral head translation observed in the MIRCT condition was significantly reduced when tensioning the biceps tendon at 30° of glenohumeral abduction compared to 0° for all tenodesis locations (P≤0.043). Tenodesis location also significantly influenced posterior humeral head translation (P=0.001), with middle and posterior positions restoring near normal humeral head position when fixed at 30° glenohumeral abduction. All SCR techniques using the biceps tendon improved the functional abduction force relative to the MIRCT condition, although no statistically significant differences were observed relative to the intact condition (P≥0.448). DiscussionSCR using the long head biceps tendon is biomechanically effective in reducing posterosuperior translation of the humeral head in the setting of a MIRCT. Graft tensioning and fixation at 30° of glenohumeral abduction combined with either a middle or posterior tenodesis location on the greater tuberosity most effectively restores near normal time-zero humeral head kinematics.

Enzymatic characterization and dominant sites of foot-and-mouth disease virus 2C protein

Foot-and-mouth disease virus (FMDV) 2C protein is a conserved non-structural protein and crucial for replication of the virus. In this study, FMDV 2C protein was prepared and the enzymatic activities were investigated in detail. The protein could digest ssDNA or ssRNA into a small fragment at about 10 nt, indicating that the protein has nuclease activity. But it did not show digestion to blunt-end dsDNA or dsRNA. The nuclease activity of 2C protein could be inhibited in 2 mM Zn2+ or Ca2+ while enhanced by Mg2+ or Mn2+. FMDV 2C protein exhibited unwinding activity to all the three kinds of dsDNA and dsRNA (5′ protruded, 3′ protruded, and blunt-end). The unwinding velocity to 5′ protruded dsRNA was higher than to the blunt-end dsRNA. 2C protein only showed unwinding activity in high concentration of Mg2+, but no unwinding activity in physiological concentrations of Mg2+ and Ca2+, as well as in cell lysate. The 2C protein could catalyze two structured ssRNA to form double strand, thus it was proved to have RNA chaperone activity. The Mg2+ and ATP in different concentrations did not show promotion to the RNA chaperone activity. Finally, six mutant proteins (K116A, D160A, D170A, N207A, R226A, and F316A) were constructed and the enzymatic activities were analyzed. All the six mutations reduced the ATPase activity, D170A and F361A could inactivate the nuclease activity, while the N207A and F316A could inactivate the helicase activity. Our study provides a comprehensive understanding of the enzymatic activities of FMDV 2C protein.

How does the polymerase of non-segmented negative strand RNA viruses commit to transcription or genome replication?

The Mononegavirales, or non-segmented negative-sense RNA viruses (nsNSVs), includes significant human pathogens, such as respiratory syncytial virus, parainfluenza virus, measles virus, Ebola virus, and rabies virus. Although these viruses differ widely in their pathogenic properties, they are united by each having a genome consisting of a single strand of negative-sense RNA. Consistent with their shared genome structure, the nsNSVs have evolved similar ways to transcribe their genome into mRNAs and replicate it to produce new genomes. Importantly, both mRNA transcription and genome replication are performed by a single virus-encoded polymerase. A fundamental and intriguing question is: how does the nsNSV polymerase commit to being either an mRNA transcriptase or a replicase? The polymerase must become committed to one process or the other either before it interacts with the genome template or in its initial interactions with the promoter sequence at the 3´ end of the genomic RNA. This review examines the biochemical, molecular biology, and structural biology data regarding the first steps of transcription and RNA replication that have been gathered over several decades for different families of nsNSVs. These findings are discussed in relation to possible models that could explain how an nsNSV polymerase initiates and commits to either transcription or genome replication.

Using AuNPs-DNA Walker with Fluorophores Detects the Hepatitis Virus Rapidly.

Viral hepatitis is a systemic infectious diseases caused by various hepatitis viruses, primarily leading to liver damage. It is widely prevalent worldwide, with hepatitis viruses categorized into five types: hepatitis A, B, C, D, and E, based on their etiology. Currently, the detection of hepatitis viruses relies on methods such as enzyme-linked immunosorbent assay (ELISA), immunoelectron microscopy to observe and identify viral particles, and in situ hybridization to detect viral DNA in tissues. However, these methods have limitations, including low sensitivity, high error rates in results, and potential false negative reactions due to occult serum infection conditions. To address these challenges, we have designed an AuNPs-DNA walker method that uses gold nanoparticles (AuNPs) and complementary DNA strands for detecting viral DNA fragments through a colorimetric assay and fluorescence detection. The DNA walker, attached to gold nanoparticles, comprises a long walking strand with a probe sequence bound and stem-loop structural strands featuring a modified fluorescent molecule at the 3' end, which contains the DNAzyme structural domain. Upon the addition of virus fragments, the target sequence binds to the probe chains. Subsequently, the long walking strand is released and continuously hybridizes with the stem-loop structural strand. The DNAzyme undergoes hydrolytical cleavage by Mg2+, breaking the stem-loop structural strand into linear single strands. As a result of these structural changes, the negative charge density in the solution decreases, weakening spatial repulsion and rapidly reducing the stability of the DNA walker. This leads to aggregation upon the addition of a high-salt solution, accompanied by a color change. Virus typing can be performed through fluorescence detection. The innovative method can detect DNA/RNA fragments with high specificity for the target sequence, reaching concentrations as low as 1 nM. Overall, our approach offers a more convenient and reliable method for the detection of hepatitis viruses.

Mechanistic Insights into How the Single Point Mutation Change the Autoantibody Repertoire.

A recent study showed that just one point mutation F33 to Y in the complementarity-determining region 1 of heavy chain (H-CDR1) could lead to the auto-antibody losing its DNA binding ability. However, the potential molecular mechanisms have not been well elucidated. In this study, we investigated how the antibody lost the DNA binding ability caused by mutation F33 to Y in the H-CDR1. We found that the electrostatic force was not the primary driving force for the interaction between anti-DNA antibodies and the antigen single strand DNA (ssDNA), and that the H-CDR2 largely contributed to the binding of antigen ssDNA, even larger than H-CDR1. The H-F33Y mutation could increase the hydrogen-bond interaction but impair the pi-pi stacking interaction between the antibody and ssDNA. We further found that F33H, W98H and Y95L in the wiletype antibody could form the stable pi-pi stacking interaction with the nucleotide bases of ssDNA. However, the Y33 in mutant could not form the parallel sandwich pi-pi stacking interaction with the ssDNA. To further confirm the importance of pi-pi stacking, the wildtype antibody and the mutants (F33YH, F33AH, W98AH and Y95AL) were experimentally expressed in CHO cells and purified, and the results from ELISA clearly showed that all the mutants lost the ssDNA binding ability. Taken together, our findings may not only deepen the understanding of the underlying interaction mechanism between autoantibody and antigen, but also broad implications in the field of antibody engineer.

Comparison of Double-Stranded DNA at the 5' and 3' Ends of the G-Triplex and Its Application in the Detection of Hg(II).

Leveraging the fluorescence enhancement effect of the G-triplex (G3)/thioflavin T (ThT) catalyzed by the adjacent double-stranded DNA positioned at the 5' terminus of the G3, the G3-specific oligonucleotide (G3MB6) was utilized to facilitate the rapid detection of mercury (Hg(II)) through thymine-Hg(II)-thymine (T-Hg(II)-T) interactions. G3MB6 adopted a hairpin structure in which partially complementary strands could be disrupted with the presence of Hg(II). It prompted the formation of double-stranded DNA by T-Hg(II)-T, inducing the unbound single strand of G3MB6 to spontaneously form a parallel G3 structure, producing a solid fluorescence signal by ThT. Conversely, fluorescence was absent without Hg(II), since no double strand and formation of G3 occurred. The fluorescence intensity of G3MB6 exhibited a positive correlation with Hg(II) concentrations from 17.72 to 300 nM (R2 = 0.9954), boasting a notably low quality of limitation (LOQ) of 17.72 nM. Additionally, it demonstrated remarkable selectivity for detecting Hg(II). Upon application to detect Hg(II) in milk samples, the recovery rates went from 100.3% to 103.2%.

Folding a Molecular Strand into a Trefoil Knot of Single Handedness with Co(II)/Co(III) Chaperones.

We report the synthesis of a right-handed (Δ-stereochemistry of strand crossings) trefoil knot from a single molecular strand containing three pyrazine-2,5-dicarboxamide units adjacent to point-chiral centers and six pyridine moieties. The oligomeric ligand strand folds into an overhand (open-trefoil) knot through the assistance of coordinatively dynamic Co(II) "chaperones" that drive the formation of a three-metal-ion circular helicate. The entangled structure is kinetically locked by oxidation to Co(III) and covalently captured by ring-closing olefin metathesis to generate a trefoil knot of single topological handedness. The stereochemistry of the strand crossings in the metal-coordinated overhand knot is governed by the stereochemistry of the point-chiral carbon centers in the ligand strand. The overhand and trefoil knots were characterized by NMR spectroscopy, mass spectrometry, and X-ray crystallography. Removal of the metal ions from the knot, followed by hydrogenation of the alkene, yielded the wholly organic trefoil knot. The metal-free knot and parent ligand were investigated by circular dichroism (CD) spectroscopy. The CD spectra indicate that the topological stereochemistry of the knot has a greater effect on the asymmetry of the chromophore environment than do the point-chiral centers of the strand.

Senataxin mediates R-loop resolution on HPV episomes.

Three-stranded DNA-RNA structures known as R-loops that form during papillomavirus transcription can cause transcription-replication conflicts and lead to DNA damage. We found that R-loops accumulated at the viral early promoter in human papillomavirus (HPV) episomal cells but were greatly reduced in cells with integrated HPV genomes. RNA-DNA helicases unwind R-loops and allow for transcription and replication to proceed. Depletion of the RNA-DNA helicase senataxin (SETX) using siRNAs increased the presence of R-loops at the viral early promoter in HPV-31 (CIN612) and HPV-16 (W12) episomal HPV cell lines. Depletion of SETX reduced viral transcripts in episomal HPV cell lines. The viral E2 protein, which binds with high affinity to specific palindromes near the promoter and origin, complexes with SETX, and both SETX and E2 are present at the viral p97 promoter in CIN612 and W12 cells. SETX overexpression increased E2 transcription activity on the p97 promoter. SETX depletion also significantly increased integration of viral genomes in CIN612 cells. Our results demonstrate that SETX resolves viral R-loops to proceed with HPV transcription and prevent genome integration.IMPORTANCEPapillomaviruses contain small circular genomes of approximately 8 kilobase pairs and undergo unidirectional transcription from the sense strand of the viral genome. Co-transcriptional R-loops were recently reported to be present at high levels in cells that maintain episomal HPV and were also detected at the early viral promoter. R-loops can inhibit transcription and DNA replication. The process that removes R-loops from the PV genome and the requisite enzymes are unknown. We propose a model in which the host RNA-DNA helicase senataxin assembles on the HPV genome to resolve R-loops in order to maintain the episomal status of the viral genome.

In vitro validation of helium ion irradiations as a function of linear energy transfer in radioresistant human malignant cells

Purpose Based on considerable interest to enlarge the experimental database of radioresistant cells after their irradiation with helium ions, HTB140, MCF-7 and HTB177 human malignant cells are exposed to helium ion beams having different linear energy transfer (LET). Materials and methods The cells are irradiated along the widened 62 MeV/u helium ion Bragg peak, providing LET of 4.9, 9.8, 23.4 and 36.8 keV/µm. Numerical simulations with the Geant4 toolkit are used for the experimental design. Cell survival is evaluated and compared with reference γ-rays. DNA double strand breaks are assessed via γ-H2AX foci. Results With the increase of LET, surviving fractions at 2 Gy decrease, while RBE (2 Gy, γ) gradually increase. For HTB140 cells, above the dose of 4 Gy, a slight saturation of survival is observed while the increase of RBE (2 Gy, γ) remains unaffected. With the increase of LET the increase of γ-H2AX foci is revealed at 0.5 h after irradiation. There is no significant difference in the number of foci between the cell lines for the same LET. From 0.5 to 24 h, the number of foci drops reaching its residual level. For each time point, there are small differences in DNA DSB among the three cell lines. Conclusion Analyses of data acquired for the three cell lines irradiated by helium ions, having different LET, reveal high elimination capacity and creation of a large number of DNA DSB with respect to γ-rays, and are between those reported for protons and carbon ions.

3D-Printed Microelectrodes for Biological Measurement.

Microelectrodes are useful electrochemical sensors that can provide spatial biological monitoring. Carbon fiber has been by far the most widely used microelectrode; however, a vast number of different materials and modification strategies have been developed to broaden the scope of microelectrodes. Carbon composite electrodes provide a simple approach to making microelectrodes with a wide range of materials, but manufacturing strategies are complex. 3D printing can provide the ability to make microelectrodes with high precision. We used fused filament fabrication to print single strands of carbon black/polylactic acid (CB/PLA) and multiwall carbon nanotube/polylactic acid (MWCNT/PLA), which were then made into microelectrodes. Microelectrodes ranged from 70 μm in diameter to 400 μm in diameter and were assessed using standard redox probes. MWCNT/PLA electrodes exhibited greater sensitivity, a lower limit of detection, and stability for the measurement of serotonin (5-HT). Both CB/PLA and MWCNT/PLA microelectrodes were able to monitor 5-HT overflow from the ex vivo ileum tissue. MWCNT/PLA microelectrodes were utilized to show differences in 5-HT overflow from ex vivo ileum and colon following exposure to odorants present in spices. These findings highlight that any conductive thermoplastic material can be fabricated into a microelectrode. This simple strategy can utilize a wide range of materials to make 3D-printed microelectrodes for a diverse range of applications.

Mechanism of Cationic Lipid Induced DNA Condensation: Lipid-DNA Coordination and Divalent Cation Charge Fluctuations.

The condensation of nucleic acids by lipids is a widespread phenomenon in biology with crucial implications for drug delivery. However, the mechanisms of DNA assembly in lipid bilayers remain insufficiently understood due to challenges in measuring and assessing each component's contribution in the lipid-DNA-cation system. This study uses all-atom molecular dynamics simulations to investigate DNA condensation in cationic lipid bilayers. Our exhaustive exploration of the thermodynamic factors reveals unique roles for phospholipid head groups and cations. We observed that bridging cations between lipid and DNA drastically reduce charges, while mobile magnesium cations "ping-ponging" between double strands create charge fluctuations. While the first factor stabilizes the DNA-lipid complex, the latter creates attractive forces to induce the spontaneous condensation of DNAs. This novel mechanism not only sheds light on the current data regarding cationic lipid-induced DNA condensation but also provides potential design strategies for creating efficient gene delivery vectors for drug delivery.

Nucleoporins cooperate with Polycomb silencers to promote transcriptional repression and repair at DNA double strand breaks.

DNA Double-strand breaks (DSBs) are harmful lesions and major sources of genomic instability. Studies have suggested that DSBs induce local transcriptional silencing that consequently promotes genomic stability. Several factors have been proposed to actively participate in this process, including ATM and Polycomb repressive complex 1 (PRC1). Here we found that disrupting PRC1 clustering disrupts DSB-induced gene silencing. Interactome analysis of PHC2, a PRC1 subunit that promotes the formation of the Polycomb body, found several nucleoporins that constitute the Nuclear Pore Complex (NPC). Similar to PHC2, depleting the nucleoporins also disrupted the DSB-induced gene silencing. We found that some of these nucleoporins, such as NUP107 and NUP43, which are members of the Y-complex of NPC, localize to DSB sites. These nucleoporin-enriched DSBs were distant from the nuclear periphery. The presence of nucleoporins and PHC2 at DSB regions were inter-dependent, suggesting that they act cooperatively in the DSB-induced gene silencing. We further found two structural components within NUP107 to be necessary for the transcriptional repression at DSBs: ATM/ATR-mediated phosphorylation at Serine37 residue within the N-terminal disordered tail, and the NUP133-binding surface at the C-terminus. These results provide a new functional interplay among nucleoporins, ATM and the Polycomb proteins in the DSB metabolism, and underscore their emerging roles in genome stability maintenance. *Hongseon Song, Yubin Bae, Sangin Kim, and Dante Deascanis contributed equally to this work.

DSB-induced oxidative stress: Uncovering crosstalk between DNA damage response and cellular metabolism

While that ROS causes DNA damage is well documented, there has been limited investigation into whether DNA damages and their repair processes can conversely induce oxidative stress. By generating a site-specific DNA double strand break (DSB) via I-SceI endonuclease expression in S. cerevisiae without damaging other cellular components, this study demonstrated that DNA repair does trigger oxidative stress. Deleting genes participating in the initiation of the resection step of homologous recombination (HR), like the MRX complex, resulted in stimulation of ROS. In contrast, deleting genes acting downstream of HR resection suppressed ROS levels. Additionally, blocking non-homologous end joining (NHEJ) also suppressed ROS. Further analysis identified Rad53 as a key player that relays DNA damage signals to alter redox metabolism in an HR-specific manner. These results suggest both HR and NHEJ can drive metabolism changes and oxidative stress, with NHEJ playing a more prominent role in ROS stimulation. Further analysis revealed a correlation between DSB-induced ROS increase and enhanced activity of NADPH oxidase Yno1 and various antioxidant enzymes. Deleting the antioxidant gene SOD1 induced synthetic lethality in HR-deficient mutants like mre11Δ and rad51Δ upon DSB induction. These findings uncover a significant interplay between DNA repair mechanisms and cellular metabolism, providing insights into understanding the side effects of genotoxic therapies and potentially aiding development of more effective cancer treatment strategies.

Smooth muscle expression of RNA editing enzyme ADAR1 controls vascular integrity and progression of atherosclerosis.

Mapping the genomic architecture of complex disease has been predicated on the understanding that genetic variants influence disease risk through modifying gene expression. However, recent discoveries have revealed that a significant burden of disease heritability in common autoinflammatory disorders and coronary artery disease is mediated through genetic variation modifying post-transcriptional modification of RNA through adenosine-to-inosine (A-to-I) RNA editing. This common RNA modification is catalyzed by ADAR enzymes, where ADAR1 edits specific immunogenic double stranded RNA (dsRNA) to prevent activation of the double strand RNA (dsRNA) sensor MDA5 ( IFIH1 ) and stimulation of an interferon stimulated gene (ISG) response. Multiple lines of human genetic data indicate impaired RNA editing and increased dsRNA sensing to be an important mechanism of coronary artery disease (CAD) risk. Here, we provide a crucial link between observations in human genetics and mechanistic cell biology leading to progression of CAD. Through analysis of human atherosclerotic plaque, we implicate the vascular smooth muscle cell (SMC) to have a unique requirement for RNA editing, and that ISG induction occurs in SMC phenotypic modulation, implicating MDA5 activation. Through culture of human coronary artery SMCs, generation of a conditional SMC specific Adar1 deletion mouse model on a pro-atherosclerosis background, and with incorporation of single cell RNA sequencing cellular profiling, we further show that Adar1 controls SMC phenotypic state, is required to maintain vascular integrity, and controls progression of atherosclerosis and vascular calcification. Through this work, we describe a fundamental mechanism of CAD, where cell type and context specific RNA editing and sensing of dsRNA mediates disease progression, bridging our understanding of human genetics and disease causality.

Theoretical Insights into MutY Glycosylase DNA Repair Mechanism.

Maintaining the integrity of the genome is fundamental to living organisms. To this end, nature developed several mechanisms to find and promptly repair DNA lesions. Among them, base excision repair (BER) enzymes evolved to efficiently carry out this task. Notably, the mechanisms allowing these proteins to search for, detect, and fix DNA damage on a biologically relevant time scale still remain partially unclear. By taking MutY, a BER enzyme implied in the repair of the 8-oxoguanine-adenine mismatches, as a model system, we shed some light on the repair mechanism through a theoretical-computational approach. First, we estimated the effect of the oxidation state of the MutY iron-sulfur cluster on the protein-DNA binding. Then, the redox thermodynamics of both the protein cluster and DNA nucleobases are calculated. Finally, the charge migration kinetics along the double strand bound to the enzyme has been evaluated. The rationalization of our results indicates that the search for DNA lesions is essentially dictated by the redox chemistry of the species involved, i.e., the iron-sulfur redox cofactor and the DNA bound to the enzyme.

Phase Separation Modulates the Thermodynamics and Kinetics of RNA Hybridization.

Biomolecular condensates can influence cellular function in a number of ways, including by changing the structural dynamics and conformational equilibria of the molecules partitioned within them. Here we use methyl transverse relaxation optimized spectroscopy (methyl-TROSY) NMR in conjunction with 2'-O-methyl labeling of RNA to characterize the thermodynamics and kinetics of RNA-RNA base pairing in condensates formed by the C-terminal intrinsically disordered region of CAPRIN1, an RNA-binding protein involved in RNA transport, translation, and stability. CAPRIN1 condensates destabilize RNA-RNA base pairing, resulting from a ∼270-fold decrease and a concomitant ∼15-fold increase in the on- and off-rates for duplex formation, respectively. The ∼30-fold slower diffusion of RNA single strands within the condensed phase partially accounts for the reduced on-rate, but the further ∼9-fold reduction likely reflects shedding of CAPRIN1 chains that are interacting with the RNA prior to hybridization. Our study emphasizes the important role of protein solvation in modulating nucleic acid recognition processes inside condensates.

DNA-PK: A synopsis beyond synapsis

Given its central role in life, DNA is remarkably easy to damage. Double strand breaks (DSBs) are the most toxic form of DNA damage, and DSBs pose the greatest danger to genomic integrity. In higher vertebrates, the non-homologous end joining pathway (NHEJ) is the predominate pathway that repairs DSBs. NHEJ has three steps: 1) DNA end recognition by the DNA dependent protein kinase [DNA-PK], 2) DNA end-processing by numerous NHEJ accessory factors, and 3) DNA end ligation by the DNA ligase IV complex (LX4). Although this would appear to be a relatively simple mechanism, it has become increasingly apparent that it is not.Recently, much insight has been derived regarding the mechanism of non-homologous end joining through a proliferation of cryo-EM studies, structure-function mutational experiments informed by these new structural data, and novel single-molecule imaging approaches. An emerging consensus in the field is that NHEJ progresses from initial DSB end recognition by DNA-PK to synapsis of the two DNA ends in a long-range synaptic complex where ends are held too far apart (115 Å) for ligation, and then progress to a short-range synaptic complex where ends are positioned close enough for ligation. What was surprising from these structural studies was the observation of two distinct types of DNA-PK dimers that represent NHEJ long-range complexes. In this review, we summarize current knowledge about the function of the distinct NHEJ synaptic complexes and align this new information with emerging cellular single-molecule microscopy studies as well as with previous studies of DNA-PK’s function in repair.

Evolution of RNA Viruses: Reasons for the Existence of Separate Plus, Minus, and Double-Strand Replication Strategies.

Plus, minus, and double-strand RNA viruses are all found in nature. We use computational models to study the relative success of these strategies. We consider translation, replication, and virion assembly inside one cell, and transmission of virions between cells. For viruses which do not incorporate a polymerase in the capsid, transmission of only plus strands is the default strategy because virions containing minus strands are not infectious. Packaging only plus strands has a significant advantage if the number of RNA strands produced per cell is larger than the number of capsids. In this case, by not packaging minus strands, the virus produces more plus-strand virions. Therefore, plus-strand viruses are selected at low multiplicity of infection. However, at high multiplicity of infection, it is preferable to package both strands because the additional minus virions produced are helpful when there are multiple infections per cell. The fact that plus-strand viruses are widespread while viruses that package both strands are not seen in nature suggests that RNA strands are indeed produced in excess over capsids, and that the multiplicity of infection is not sufficiently high to favor the production of both kinds of virions. For double-strand viruses, we show that it is advantageous to produce only plus strands from the double strand within the cell, as is observed in real viruses. The reason for the success of minus-strand viruses is more puzzling initially. For viruses that incorporate a polymerase in the virion, minus virions are infectious. However, this is not sufficient to explain the success of minus-strand viruses, because in this case, viruses that package both strands outcompete those that package only minus or only plus. Real minus-strand viruses make use of replicable strands that are coated by a nucleoprotein, and separate translatable plus strands that are uncoated. Here we show that when there are distinct replicable and translatable strands, minus-strand viruses are selected.