Electrophoretic Mobility Assay to Separate Supercoiled, Catenated, and Knotted DNA Molecules.

Two-dimensional (2D) agarose gel electrophoresis is the method of choice to analyze DNA topology. The possibility to use E. coli strains with different genetic backgrounds in combination with nicking enzymes and different concentrations of norfloxacin improves the resolution of 2D gels to study the electrophoretic behavior of three different families of DNA topoisomers: supercoiled DNA molecules, post-replicative catenanes, and knotted DNA molecules. Here, we describe the materials and procedures required to optimize their separation by 2D gels. Understanding the differences in their electrophoretic behavior can help explain some important physical characteristics of these different types of DNA topoisomers. Key features • Preparative method to enrich DNA samples of supercoiled, catenated, and knotted families of topoisomers, later analyzed by 2D gels (or other techniques, e.g., microscopy). • 2D gels facilitate the separation of the topoisomers of any given circular DNA molecule. • Separation of DNA molecules with the same molecular masses but different shapes can be optimized by modifying the conditions of 2D gels. • Evaluating the roles of electric field and agarose concentration on the electrophoretic mobility of DNA topoisomers sheds light on their physical characteristics.

Are serious games an alternative to traditional personality questionnaires? Initial analysis of a gamified assessment.

Personality questionnaires stand as crucial instruments in personnel selection but their limitations turn the interest towards alternatives like game-related assessments (GRAs). GRAs developed for goals other than fun are called serious games. Within them, gamified assessments are serious games that share similarities with traditional assessments (questionnaires, situational judgment tests, etc.) but they incorporate game elements like story, music, and game dynamics. This paper aims to contribute to the research on serious games as an alternative to traditional personality questionnaires by analyzing the characteristics of a gamified assessment called VASSIP. This gamified assessment, based on an existing measure of the Big Five personality traits, incorporates game elements such as storyfication, immersion, and non-evaluable gamified dynamics. The study performed included 98 university students (77.6% with job experience) as participants. They completed the original personality measure (BFI-2-S), the gamified evaluation of personality (VASSIP), a self-report measure of the main dimensions of job performance (task performance, contextual performance, and counterproductive work behaviors), and measures of applicant reactions to BFI-2-S and VASSIP. Results showed that the gamified assessment behaved similarly to the original personality measure in terms of reliability and participants' scores, although the scores in Conscientiousness were substantially higher in VASSIP. Focusing on self-reports of the three dimensions of job performance, regression models showed that the gamified assessment could explain all of them. Regarding applicant reactions, the gamified assessment obtained higher scores in perceptions of comfort, predictive validity, and attractiveness, although the effect size was small except for the latter. Finally, all applicant reactions except for attractiveness were related to age and personality traits. In conclusion, gamified assessments have the potential to be an alternative to traditional personality questionnaires but VASSIP needs more research before its application in actual selection processes.

Variable Penetrance and Expressivity of a Rare Pore Loss-of-Function Mutation (p.L889V) of Nav1.5 Channels in Three Spanish Families.

A novel rare mutation in the pore region of Nav1.5 channels (p.L889V) has been found in three unrelated Spanish families that produces quite diverse phenotypic manifestations (Brugada syndrome, conduction disease, dilated cardiomyopathy, sinus node dysfunction, etc.) with variable penetrance among families. We clinically characterized the carriers and recorded the Na+ current (INa) generated by p.L889V and native (WT) Nav1.5 channels, alone or in combination, to obtain further insight into the genotypic-phenotypic relationships in patients carrying SCN5A mutations and in the molecular determinants of the Nav1.5 channel function. The variant produced a strong dominant negative effect (DNE) since the peak INa generated by p.L889V channels expressed in Chinese hamster ovary cells, either alone (-69.4 ± 9.0 pA/pF) or in combination with WT (-62.2 ± 14.6 pA/pF), was significantly (n ≥ 17, p < 0.05) reduced compared to that generated by WT channels alone (-199.1 ± 44.1 pA/pF). The mutation shifted the voltage dependence of channel activation and inactivation to depolarized potentials, did not modify the density of the late component of INa, slightly decreased the peak window current, accelerated the recovery from fast and slow inactivation, and slowed the induction kinetics of slow inactivation, decreasing the fraction of channels entering this inactivated state. The membrane expression of p.L889V channels was low, and in silico molecular experiments demonstrated profound alterations in the disposition of the pore region of the mutated channels. Despite the mutation producing a marked DNE and reduction in the INa and being located in a critical domain of the channel, its penetrance and expressivity are quite variable among the carriers. Our results reinforce the argument that the incomplete penetrance and phenotypic variability of SCN5A loss-of-function mutations are the result of a combination of multiple factors, making it difficult to predict their expressivity in the carriers despite the combination of clinical, genetic, and functional studies.

Coexistent HCN4 and GATA5 rare variants and Atrial Fibrillation in a large Spanish Family.

BACKGROUND: Familial association of atrial fibrillation (AF) can involve single gene variants related to known arrhythmogenic mechanisms; however, genome-wide association studies often disclose complex genetic variants in familial and non-familial AF, making it difficult to relate to known pathogenetic mechanisms. METHODS: The finding of 4 siblings with AF led to studying 47 members of a family. Long-term Holter monitoring (298 hours average) ruled out silent AFWhole-exome sequencing was performed and variants shared by the index cases were filtered and prioritized according to current recommendations. HCN4 currents (IHCN4) were recorded in Chinese hamster ovary cells expressing human p.P1163H and/or native Hcn4 channels using the patch-clamp technique and topologically associated domain analysis of GATA5 variant carriers were performed. RESULTS: The clinical study diagnosed 2 more AF cases. Five family members carried the heterozygous p.P1163H, HCN4 variant, 14 the intronic 20,61040536,G,A GATA5 rare variant, and 9 carried both variants (HCN4+GATA5). Five of the 6 AF cases (onset age ranging 33-70 years) carried both variants and one the GATA5 variant alone. Multivariate analysis showed that the presence of HCN4+GATA5 variants significantly and independently increased AF risk [OR=32.740 (1.812-591.408)] and not age, hypertension or overweight. Functional testing showed that IHcn4 generated by heterozygous p.P1163H were normal. Topologically associating domain analysis suggested that GATA5 could affect the expression of many genes, including those encoding microRNA-1. CONCLUSION: The coincidence of two rare gene variants was independently associated with AF, but functional studies do not allow the postulation of the arrhythmogenic mechanism(s) involved.

A gain-of-function HCN4 mutant in the HCN domain is responsible for inappropriate sinus tachycardia in a Spanish family.

In a family with inappropriate sinus tachycardia (IST), we identified a mutation (p.V240M) of the hyperpolarization-activated cyclic nucleotide-gated type 4 (HCN4) channel, which contributes to the pacemaker current (If) in human sinoatrial node cells. Here, we clinically study fifteen family members and functionally analyze the p.V240M variant. Macroscopic (IHCN4) and single-channel currents were recorded using patch-clamp in cells expressing human native (WT) and/or p.V240M HCN4 channels. All p.V240M mutation carriers exhibited IST that was accompanied by cardiomyopathy in adults. IHCN4 generated by p.V240M channels either alone or in combination with WT was significantly greater than that generated by WT channels alone. The variant, which lies in the N-terminal HCN domain, increased the single-channel conductance and opening frequency and probability of HCN4 channels. Conversely, it did not modify the channel sensitivity for cAMP and ivabradine or the level of expression at the membrane. Treatment with ivabradine based on functional data reversed the IST and the cardiomyopathy of the carriers. In computer simulations, the p.V240M gain-of-function variant increases If and beating rate and thus explains the IST of the carriers. The results demonstrate the importance of the unique HCN domain in HCN4, which stabilizes the channels in the closed state.

Large-scale expansions of Friedreich's ataxia GAA•TTC repeats in an experimental human system: role of DNA replication and prevention by LNA-DNA oligonucleotides and PNA oligomers.

Friedreich's ataxia (FRDA) is caused by expansions of GAA•TTC repeats in the first intron of the human FXN gene that occur during both intergenerational transmissions and in somatic cells. Here we describe an experimental system to analyze large-scale repeat expansions in cultured human cells. It employs a shuttle plasmid that can replicate from the SV40 origin in human cells or be stably maintained in S. cerevisiae utilizing ARS4-CEN6. It also contains a selectable cassette allowing us to detect repeat expansions that accumulated in human cells upon plasmid transformation into yeast. We indeed observed massive expansions of GAA•TTC repeats, making it the first genetically tractable experimental system to study large-scale repeat expansions in human cells. Further, GAA•TTC repeats stall replication fork progression, while the frequency of repeat expansions appears to depend on proteins implicated in replication fork stalling, reversal, and restart. Locked nucleic acid (LNA)-DNA mixmer oligonucleotides and peptide nucleic acid (PNA) oligomers, which interfere with triplex formation at GAA•TTC repeats in vitro, prevented the expansion of these repeats in human cells. We hypothesize, therefore, that triplex formation by GAA•TTC repeats stall replication fork progression, ultimately leading to repeat expansions during replication fork restart.

A Cantú syndrome mutation produces dual effects on KATP channels by disrupting ankyrin B regulation.

ATP-sensitive potassium (KATP) channels composed of Kir6.x and sulfonylurea receptor (SURs) subunits couple cellular metabolism to electrical activity. Cantú syndrome (CS) is a rare disease caused by mutations in the genes encoding Kir6.1 (KCNJ8) and SUR2A (ABCC9) that produce KATP channel hyperactivity due to a reduced channel block by physiological ATP concentrations. We functionally characterized the p.S1054Y SUR2A mutation identified in two CS carriers, who exhibited a mild phenotype although the mutation was predicted as highly pathogenic. We recorded macroscopic and single-channel currents in CHO and HEK-293 cells and measured the membrane expression of the channel subunits by biotinylation assays in HEK-293 cells. The mutation increased basal whole-cell current density and at the single-channel level, it augmented opening frequency, slope conductance, and open probability (Po), and promoted the appearance of multiple conductance levels. p.S1054Y also reduced Kir6.2 and SUR2A expression specifically at the membrane. Overexpression of ankyrin B (AnkB) prevented these gain- and loss-of-function effects, as well as the p.S1054Y-induced reduction of ATP inhibition of currents measured in inside-out macropatches. Yeast two-hybrid assays suggested that SUR2A WT and AnkB interact, while p.S1054Y interaction with AnkB is decreased. The p.E322K Kir6.2 mutation, which prevents AnkB binding to Kir6.2, produced similar biophysical alterations than p.S1054Y. Our results are the first demonstration of a CS mutation whose functional consequences involve the disruption of AnkB effects on KATP channels providing a novel mechanism by which CS mutations can reduce ATP block. Furthermore, they may help explain the mild phenotype associated with this mutation.

Tbx5 variants disrupt Nav1.5 function differently in patients diagnosed with Brugada or Long QT Syndrome.

AIMS: The transcription factor Tbx5 controls cardiogenesis and drives Scn5a expression in mice. We have identified two variants in TBX5 encoding p. D111Y and p. F206L Tbx5, respectively, in two unrelated patients with structurally normal hearts diagnosed with long QT (LQTS) and Brugada (BrS) syndrome. Here, we characterized the consequences of each variant to unravel the underlying disease mechanisms. METHODS AND RESULTS: We combined clinical analysis with in vivo and in vitro electrophysiological and molecular techniques in human-induced pluripotent stem-cell-derived cardiomyocytes (hiPSC-CMs), HL-1 cells, and cardiomyocytes from mice trans-expressing human wild-type (WT) or mutant proteins. Tbx5 increased transcription of SCN5A encoding cardiac Nav1.5 channels, while repressing CAMK2D and SPTBN4 genes encoding Ca/calmodulin kinase IIδ (CaMKIIδ) and ßIV-spectrin, respectively. These effects significantly increased Na current (INa) in hiPSC-CMs and in cardiomyocytes from mice trans-expressing Tbx5. Consequently, action potential (AP) amplitudes increased and QRS interval narrowed in the mouse electrocardiogram. p. F206L Tbx5 bound to the SCN5A promoter failed to transactivate it, thus precluding the pro-transcriptional effect of WT Tbx5. Therefore, p. F206L markedly decreased INa in hiPSC-CM, HL-1 cells and mouse cardiomyocytes. The INa decrease in p. F206L trans-expressing mice translated into QRS widening and increased flecainide sensitivity. p. D111Y Tbx5 increased SCN5A expression but failed to repress CAMK2D and SPTBN4. The increased CaMKIIδ and ßIV-spectrin significantly augmented the late component of INa (INaL) which, in turn, significantly prolonged AP duration in both hiPSC-CMs and mouse cardiomyocytes. Ranolazine, a selective INaL inhibitor, eliminated the QT and QTc intervals prolongation seen in p. D111Y trans-expressing mice. CONCLUSIONS: In addition to peak INa, Tbx5 critically regulates INaL and the duration of repolarization in human cardiomyocytes. Our original results suggest that TBX5 variants associate with and modulate the intensity of the electrical phenotype in LQTS and BrS patients.

Zfhx3 Transcription Factor Represses the Expression of SCN5A Gene and Decreases Sodium Current Density (INa).

The ZFHX3 and SCN5A genes encode the zinc finger homeobox 3 (Zfhx3) transcription factor (TF) and the human cardiac Na+ channel (Nav1.5), respectively. The effects of Zfhx3 on the expression of the Nav1.5 channel, and in cardiac excitability, are currently unknown. Additionally, we identified three Zfhx3 variants in probands diagnosed with familial atrial fibrillation (p.M1260T) and Brugada Syndrome (p.V949I and p.Q2564R). Here, we analyzed the effects of native (WT) and mutated Zfhx3 on Na+ current (INa) recorded in HL-1 cardiomyocytes. ZFHX3 mRNA can be detected in human atrial and ventricular samples. In HL-1 cardiomyocytes, transfection of Zfhx3 strongly reduced peak INa density, while the silencing of endogenous expression augmented it (from -65.9 ± 8.9 to -104.6 ± 10.8 pA/pF; n ≥ 8, p < 0.05). Zfhx3 significantly reduced the transcriptional activity of human SCN5A, PITX2, TBX5, and NKX25 minimal promoters. Consequently, the mRNA and/or protein expression levels of Nav1.5 and Tbx5 were diminished (n ≥ 6, p < 0.05). Zfhx3 also increased the expression of Nedd4-2 ubiquitin-protein ligase, enhancing Nav1.5 proteasomal degradation. p.V949I, p.M1260T, and p.Q2564R Zfhx3 produced similar effects on INa density and time- and voltage-dependent properties in WT. WT Zfhx3 inhibits INa as a result of a direct repressor effect on the SCN5A promoter, the modulation of Tbx5 increasing on the INa, and the increased expression of Nedd4-2. We propose that this TF participates in the control of cardiac excitability in human adult cardiac tissue.

Rad9-mediated checkpoint activation is responsible for elevated expansions of GAA repeats in CST-deficient yeast.

Large-scale expansion of (GAA)n repeats in the first intron of the FXN gene is responsible for the severe neurodegenerative disease, Friedreich's ataxia in humans. We have previously conducted an unbiased genetic screen for GAA repeat instability in a yeast experimental system. The majority of genes that came from this screen encoded the components of DNA replication machinery, strongly implying that replication irregularities are at the heart of GAA repeat expansions. This screen, however, also produced two unexpected hits: members of the CST complex, CDC13 and TEN1 genes, which are required for telomere maintenance. To understand how the CST complex could affect intra-chromosomal GAA repeats, we studied the well-characterized temperature-sensitive cdc13-1 mutation and its effects on GAA repeat instability in yeast. We found that in-line with the screen results, this mutation leads to ∼10-fold increase in the rate of large-scale expansions of the (GAA)100 repeat at semi-permissive temperature. Unexpectedly, the hyper-expansion phenotype of the cdc13-1 mutant largely depends on activation of the G2/M checkpoint, as deletions of individual genes RAD9, MEC1, RAD53, and EXO1 belonging to this pathway rescued the increased GAA expansions. Furthermore, the hyper-expansion phenotype of the cdc13-1 mutant depended on the subunit of DNA polymerase δ, Pol32. We hypothesize, therefore, that increased repeat expansions in the cdc13-1 mutant happen during post-replicative repair of nicks or small gaps within repetitive tracts during the G2 phase of the cell cycle upon activation of the G2/M checkpoint.

Two-Dimensional Gel Electrophoresis to Study the Activity of Type IIA Topoisomerases on Plasmid Replication Intermediates.

DNA topoisomerases are the enzymes that regulate DNA topology in all living cells. Since the discovery and purification of ω (omega), when the first were topoisomerase identified, the function of many topoisomerases has been examined. However, their ability to relax supercoiling and unlink the pre-catenanes of partially replicated molecules has received little attention. Here, we used two-dimensional agarose gel electrophoresis to test the function of three type II DNA topoisomerases in vitro: the prokaryotic DNA gyrase, topoisomerase IV and the human topoisomerase 2α. We examined the proficiency of these topoisomerases on a partially replicated bacterial plasmid: pBR-TerE@AatII, with an unidirectional replicating fork, stalled when approximately half of the plasmid had been replicated in vivo. DNA was isolated from two strains of Escherichia coli: DH5αF' and parE10. These experiments allowed us to assess, for the first time, the efficiency of the topoisomerases examined to resolve supercoiling and pre-catenanes in partially replicated molecules and fully replicated catenanes formed in vivo. The results obtained revealed the preferential functions and also some redundancy in the abilities of these DNA topoisomerases in vitro.

The p.P888L SAP97 polymorphism increases the transient outward current (Ito,f) and abbreviates the action potential duration and the QT interval.

Synapse-Associated Protein 97 (SAP97) is an anchoring protein that in cardiomyocytes targets to the membrane and regulates Na+ and K+ channels. Here we compared the electrophysiological effects of native (WT) and p.P888L SAP97, a common polymorphism. Currents were recorded in cardiomyocytes from mice trans-expressing human WT or p.P888L SAP97 and in Chinese hamster ovary (CHO)-transfected cells. The duration of the action potentials and the QT interval were significantly shorter in p.P888L-SAP97 than in WT-SAP97 mice. Compared to WT, p.P888L SAP97 significantly increased the charge of the Ca-independent transient outward (Ito,f) current in cardiomyocytes and the charge crossing Kv4.3 channels in CHO cells by slowing Kv4.3 inactivation kinetics. Silencing or inhibiting Ca/calmodulin kinase II (CaMKII) abolished the p.P888L-induced Kv4.3 charge increase, which was also precluded in channels (p.S550A Kv4.3) in which the CaMKII-phosphorylation is prevented. Computational protein-protein docking predicted that p.P888L SAP97 is more likely to form a complex with CaMKII than WT. The Na+ current and the current generated by Kv1.5 channels increased similarly in WT-SAP97 and p.P888L-SAP97 cardiomyocytes, while the inward rectifier current increased in WT-SAP97 but not in p.P888L-SAP97 cardiomyocytes. The p.P888L SAP97 polymorphism increases the Ito,f, a CaMKII-dependent effect that may increase the risk of arrhythmias.

Mrc1 and Tof1 prevent fragility and instability at long CAG repeats by their fork stabilizing function.

Fork stabilization at DNA impediments is key to maintaining replication fork integrity and preventing chromosome breaks. Mrc1 and Tof1 are two known stabilizers that travel with the replication fork. In addition to a structural role, Mrc1 has a DNA damage checkpoint function. Using a yeast model system, we analyzed the role of Mrc1 and Tof1 at expanded CAG repeats of medium and long lengths, which are known to stall replication forks and cause trinucleotide expansion diseases such as Huntington's disease and myotonic dystrophy. We demonstrate that the fork stabilizer but not the checkpoint activation function of Mrc1 is key for preventing DNA breakage and death of cells containing expanded CAG tracts. In contrast, both Mrc1 functions are important in preventing repeat length instability. Mrc1 has a general fork protector role that is evident at forks traversing both repetitive and non-repetitive DNA, though it becomes crucial at long CAG repeat lengths. In contrast, the role of Tof1 in preventing fork breakage is specific to long CAG tracts of 85 or more repeats. Our results indicate that long CAG repeats have a particular need for Tof1 and highlight the importance of fork stabilizers in maintaining fork integrity during replication of structure-forming repeats.

Direct Evidence for the Formation of Precatenanes during DNA Replication.

The dynamics of DNA topology during replication are still poorly understood. Bacterial plasmids are negatively supercoiled. This underwinding facilitates strand separation of the DNA duplex during replication. Leading the replisome, a DNA helicase separates the parental strands that are to be used as templates. This strand separation causes overwinding of the duplex ahead. If this overwinding persists, it would eventually impede fork progression. In bacteria, DNA gyrase and topoisomerase IV act ahead of the fork to keep DNA underwound. However, the processivity of the DNA helicase might overcome DNA gyrase and topoisomerase IV. It was proposed that the overwinding that builds up ahead of the fork could force it to swivel and diffuse this positive supercoiling behind the fork where topoisomerase IV would also act to maintain replicating the DNA underwound. Putative intertwining of sister duplexes in the replicated region are called precatenanes. Fork swiveling and the formation of precatenanes, however, are still questioned. Here, we used classical genetics and high resolution two-dimensional agarose gel electrophoresis to examine the torsional tension of replication intermediates of three bacterial plasmids with the fork stalled at different sites before termination. The results obtained indicated that precatenanes do form as replication progresses before termination.

Electrophoretic mobility of supercoiled, catenated and knotted DNA molecules.

We systematically varied conditions of two-dimensional (2D) agarose gel electrophoresis to optimize separation of DNA topoisomers that differ either by the extent of knotting, the extent of catenation or the extent of supercoiling. To this aim we compared electrophoretic behavior of three different families of DNA topoisomers: (i) supercoiled DNA molecules, where supercoiling covered the range extending from covalently closed relaxed up to naturally supercoiled DNA molecules; (ii) postreplicative catenanes with catenation number increasing from 1 to ∼15, where both catenated rings were nicked; (iii) knotted but nicked DNA molecules with a naturally arising spectrum of knots. For better comparison, we studied topoisomer families where each member had the same total molecular mass. For knotted and supercoiled molecules, we analyzed dimeric plasmids whereas catenanes were composed of monomeric forms of the same plasmid. We observed that catenated, knotted and supercoiled families of topoisomers showed different reactions to changes of agarose concentration and voltage during electrophoresis. These differences permitted us to optimize conditions for their separation and shed light on physical characteristics of these different types of DNA topoisomers during electrophoresis.

Topoisomerase 2 is dispensable for the replication and segregation of small yeast artificial chromosomes (YACs).

DNA topoisomerases are thought to play a critical role in transcription, replication and recombination as well as in the condensation and segregation of sister duplexes during cell division. Here, we used high-resolution two-dimensional agarose gel electrophoresis to study the replication intermediates and final products of small circular and linear minichromosomes of Saccharomyces cerevisiae in the presence and absence of DNA topoisomerase 2. The results obtained confirmed that whereas for circular minichromosomes, catenated sister duplexes accumulated in the absence of topoisomerase 2, linear YACs were able to replicate and segregate regardless of this topoisomerase. The patterns of replication intermediates for circular and linear YACs displayed significant differences suggesting that DNA supercoiling might play a key role in the modulation of replication fork progression. Altogether, this data supports the notion that for linear chromosomes the torsional tension generated by transcription and replication dissipates freely throughout the telomeres.