DNA Circles Research Articles

Capillary gel electrophoresis with laser‐induced fluorescence of plasmid DNA in untreated capillary

A technique utilizing CGE-LIF in a bare capillary has been developed and evaluated for the detection of the three different topoisomers (linear, open circle, and supercoiled) of plasmid DNA along with the prospect of the dimer form of the supercoiled isoform. Utilizing the zwitterionic buffer, HEPES with boric acid sufficiently prevented capillary wall interactions and minimized the EOF, enabling a well-resolved separation of different plasmid isoforms. Multiple run conditions including buffer concentration and pH, hydroxypropylmethylcellulose size and amount, injection parameters, and the presence of an intercalating dye were evaluated and optimized. In addition, the feasibility of using this method as a platform for varying sizes of plasmid was investigated.

Segregation of yeast nuclear pores

During mitosis in Saccharomyces cerevisiae, senescence factors such as extrachromosomal ribosomal DNA circles (ERCs) are retained in the mother cell and excluded from the bud/daughter cell. Shcheprova et al. proposed a model suggesting segregation of ERCs through their association with nuclear pore complexes (NPCs) and retention of pre-existing NPCs in the mother cell during mitosis. However, this model is inconsistent with previous data and we demonstrate here that NPCs do efficiently migrate from the mother into the bud. Therefore, binding to NPCs does not seem to explain the retention of ERCs in the mother cell.

DNA ligase 4 stabilizes the ribosomal DNA array upon fork collapse at the replication fork barrier

DNA double-strand breaks (DSB) were shown to occur at the replication fork barrier in the ribosomal DNA of Saccharomyces cerevisiae using 2D-gel electrophoresis. Their origin, nature and magnitude, however, have remained elusive. We quantified these DSBs and show that a surprising 14% of replicating ribosomal DNA molecules are broken at the replication fork barrier in replicating wild-type cells. This translates into an estimated steady-state level of 7–10 DSBs per cell during S-phase. Importantly, breaks detectable in wild-type and sgs1 mutant cells differ from each other in terms of origin and repair. Breaks in wild-type, which were previously reported as DSBs, are likely an artefactual consequence of nicks nearby the rRFB. Sgs1 deficient cells, in which replication fork stability is compromised, reveal a class of DSBs that are detectable only in the presence of functional Dnl4. Under these conditions, Dnl4 also limits the formation of extrachromosomal ribosomal DNA circles. Consistently, dnl4 cells displayed altered fork structures at the replication fork barrier, leading us to propose an as yet unrecognized role for Dnl4 in the maintenance of ribosomal DNA stability.

Action at Hooked or Twisted–Hooked DNA Juxtapositions Rationalizes Unlinking Preference of Type-2 Topoisomerases

The mathematical basis of the hypothesis that type-2 topoisomerases recognize and act at specific DNA juxtapositions has been investigated by coarse-grained lattice polymer models, showing that selective segment passages at hooked juxtapositions can result in dramatic reductions in catenane and knot populations. The lattice modeling approach is here extended to account for the narrowing of variance of linking number ( Lk) of DNA circles by type-2 topoisomerases. In general, the steady-state variance of Lk resulting from selective segment passages at a specific juxtaposition geometry j is inversely proportional to the average linking number, 〈 Lk〉 j , of circles with the given juxtaposition. Based on this formulation, we demonstrate that selective segment passages at hooked juxtapositions reduce the variance of Lk. The dependence of this effect on model DNA circle size is remarkably similar to that observed experimentally for type-2 topoisomerases, which appear to be less capable in narrowing Lk variance for small DNA circles than for larger DNA circles. This behavior is rationalized by a substantial cancellation of writhe in small circles with hook-like juxtapositions. During our simulations, we uncovered a twisted variation of the hooked juxtaposition that has an even more dramatic effect on Lk variance narrowing than the hooked juxtaposition. For an extended set of juxtapositions, we detected a significant correlation between the Lk narrowing potential and the logarithmic decatenating and unknotting potentials for a given juxtaposition, a trend reminiscent of scaling relations observed with experimental measurements on type-2 topoisomerases from a variety of organisms. The consistent agreement between theory and experiment argues for type-2 topoisomerase action at hooked or twisted–hooked DNA juxtapositions.

Local site preference rationalizes disentangling by DNA topoisomerases

To rationalize the disentangling action of type II topoisomerases, an improved wormlike DNA model was used to delineate the degree of unknotting and decatenating achievable by selective segment passage at specific juxtaposition geometries and to determine how these activities were affected by DNA circle size and solution ionic strength. We found that segment passage at hooked geometries can reduce knot populations as dramatically as seen in experiments. Selective segment passage also provided theoretical underpinning for an intriguing empirical scaling relation between unknotting and decatenating potentials.

Brownian Dynamics Simulation of FCS Measurements on Single Fluorophore-Labeled Superhelical DNA

We investigated the dynamics of a single-fluorophore-labeled pUC18 plasmid through a Brownian dynamics algorithm, followed up by a simulation of the Fluorescence Correlation Spectroscopy (FCS) process. Recent experimental FCS measurements indicated a sensitivity of the monomer mean square displacements in DNA circles towards superhelicity. Simulations with homogeneous DNA elasticity and local straight equilibrium are not sufficient to reproduce this observed behavior. But inserting permanently bent sequences into the DNA, which favor end loop formation, caused a dependence of the calculated FCS correlation curves on superhelical density. Furthermore, our simulations allow us to take into account the orientation of the fluorophore in polarized excitation, which might explain the observed appearance of a Rouse-like regime at intermediate time scales.

Extrachromosomal circles of satellite repeats and 5S ribosomal DNA in human cells

BackgroundExtrachomosomal circular DNA (eccDNA) is ubiquitous in eukaryotic organisms and was detected in every organism tested, including in humans. A two-dimensional gel electrophoresis facilitates the detection of eccDNA in preparations of genomic DNA. Using this technique we have previously demonstrated that most of eccDNA consists of exact multiples of chromosomal tandemly repeated DNA, including both coding genes and satellite DNA.ResultsHere we report the occurrence of eccDNA in every tested human cell line. It has heterogeneous mass ranging from less than 2 kb to over 20 kb. We describe eccDNA homologous to human alpha satellite and the SstI mega satellite. Moreover, we show, for the first time, circular multimers of the human 5S ribosomal DNA (rDNA), similar to previous findings in Drosophila and plants. We further demonstrate structures that correspond to intermediates of rolling circle replication, which emerge from the circular multimers of 5S rDNA and SstI satellite.ConclusionsThese findings, and previous reports, support the general notion that every chromosomal tandem repeat is prone to generate eccDNA in eukryoric organisms including humans. They suggest the possible involvement of eccDNA in the length variability observed in arrays of tandem repeats. The implications of eccDNA on genome biology may include mechanisms of centromere evolution, concerted evolution and homogenization of tandem repeats and genomic plasticity.

α3, a transposable element that promotes host sexual reproduction

Theoretical models predict that selfish DNA elements require host sex to persist in a population. Therefore, a transposon that induces sex would strongly favor its own spread. We demonstrate that a protein homologous to transposases, called alpha3, was essential for mating type switch in Kluyveromyces lactis. Mutational analysis showed that amino acids conserved among transposases were essential for its function. During switching, sequences in the 5' and 3' flanking regions of the alpha3 gene were joined, forming a DNA circle, showing that alpha3 mobilized from the genome. The sequences encompassing the alpha3 gene circle junctions in the mating type alpha (MATalpha) locus were essential for switching from MATalpha to MATa, suggesting that alpha3 mobilization was a coupled event. Switching also required a DNA-binding protein, Mating type switch 1 (Mts1), whose binding sites in MATalpha were important. Expression of Mts1 was repressed in MATa/MATalpha diploids and by nutrients, limiting switching to haploids in low-nutrient conditions. A hairpin-capped DNA double-strand break (DSB) was observed in the MATa locus in mre11 mutant strains, indicating that mating type switch was induced by MAT-specific DSBs. This study provides empirical evidence for selfish DNA promoting host sexual reproduction by mediating mating type switch.

Multiplexed Detection of Site Specific Recombinase and DNA Topoisomerase Activities at the Single Molecule Level

We previously demonstrated the conversion of a single human topoisomerase I mediated DNA cleavage-ligation event happening within nanometer dimensions to a micrometer-sized DNA molecule, readily detectable using standard fluorescence microscopy. This conversion was achieved by topoisomerase I mediated closure of a nicked DNA circle followed by rolling circle amplification leading to an anchored product that was visualized at the single molecule level by hybridization to fluorescently labeled probes (Stougaard et al. ACS Nano 2009, 3, 223-33). An important inherent property of the presented setup is, at least in theory, the easy adaptability to multiplexed enzyme detection simply by using differently labeled probes for the detection of rolling circle products of different circularized substrates. In the present study we demonstrate the specific detection of three different enzyme activities, human topoisomerase I, and Flp and Cre recombinase in nuclear extracts from human cells one at a time or multiplexed using the rolling circle amplification based single-molecule detection system. Besides serving as a proof-of-principle for the feasibility of the presented assay for multiplexed enzyme detection in crude human cell extracts, the simultaneous detection of Flp and Cre activities in a single sample may find immediate practical use since these enzymes are often used in combination to control mammalian gene expression.

DNA C-circles are specific and quantifiable markers of alternative-lengthening-of-telomeres activity

Alternative lengthening of telomeres (ALT) is likely to be an important target for anticancer treatment as approximately 10% of cancers depend on this telomere maintenance mechanism for continued growth, and inhibition of ALT can cause cellular senescence. However, no ALT inhibitors have been developed for therapeutic use because of the lack of a suitable ALT activity assay and of known ALT-specific target molecules. Here we show that partially single-stranded telomeric (CCCTAA)(n) DNA circles (C-circles) are ALT specific. We provide an assay that is rapidly and linearly responsive to ALT activity and that is suitable for screening for ALT inhibitors. We detect C-circles in blood from ALT(+) osteosarcoma patients, suggesting that the C-circle assay (CC assay) may have clinical utility for diagnosis and management of ALT(+) tumors.

HIV‐infected Patients with a Large Thymus Maintain Higher CD4 Counts in a 5‐year Follow‐up Study of Patients Treated with Highly Active Antiretroviral Therapy

CD4 recovery in HIV-infected patients treated with highly active antiretroviral therapy (HAART) is in part believed to be dependent on the degree of preserved thymic function. We investigated whether the thymus has a prolonged effect on CD4 recovery. Total and naïve CD4 counts as well as thymic output determined as the number of CD4 + cells containing T-cell receptor-rearrangement excision DNA circles were measured prospectively in 25 HIV-infected patients with known thymic size during 5 years of HAART. Patients with larger thymic size had at all time points of follow-up significantly higher CD4 counts than patients with minimal thymic size (P = 0.0036). The CD4 increase from time of initiation of HAART until 6 months of follow-up differed significantly between the two thymic groups (P = 0.045), but did not at later time points. Thymic output remained significantly higher in patients with larger thymic size at follow-up. However, no difference in the increase in thymic output was seen between thymic groups. In conclusion, the importance of the thymus to the rate of cellular restoration seems primarily to lie within the first two years of HAART. However, patients with larger thymic size are able to maintain higher CD4 counts even after 5 years of HAART.

The feature of clonal expansion of TCR Vβ repertoire, thymic recent output function and TCRζ chain expression in patients with immune thrombocytopenic purpura

In order to identify the feature of T cells immune status in idiopathic/immune thrombocytopenic purpura (ITP) patients, TCR Vbeta repertoire, T-cell receptor (TCR) excision DNA circles (TRECs) and TCRzeta chain gene expression were examined. Reverse-transcriptase polymerase chain reaction, genescan and real-time PCR techniques were used to analyze. DNA and cDNA from peripheral blood mononuclear cells (PBMCs) of 18 ITP patients were investigated and 25 normal individuals served as control. The results showed that only 4-11 Vbeta subfamilies could be detected in each ITP case. The most frequently expressed Vbeta subfamilies were Vbeta2 and Vbeta3, while 11 Vbeta subfamilies were absent. Clonal expansion of Vbeta T cells were found in all patients. The most frequent clonal expansion T cell was Vbeta21. The number of TRECs in ITP patients (2.60 +/- 2.99 copies/1000 PBMCs) was not significant different, compared with control (3.76 +/- 3.42 copies/1000 PBMCs). The expression level of TCRzeta gene was significantly lower in ITP samples than those in control (P = 0.017). In summary, T cells in ITP patients were characterized by restricted expression of TCR Vbeta subfamilies and clonal expansion predominant in Vbeta21. The alteration of peripheral TCR Vbeta repertoire pattern may not relate to the thymic, recent output function in ITP patients. Our report is the first description of decreased TCRzeta mRNA level in the majority of ITP patients.

Novel circular DNA viruses in stool samples of wild-living chimpanzees

Viral particles in stool samples from wild-living chimpanzees were analysed using random PCR amplification and sequencing. Sequences encoding proteins distantly related to the replicase protein of single-stranded circular DNA viruses were identified. Inverse PCR was used to amplify and sequence multiple small circular DNA viral genomes. The viral genomes were related in size and genome organization to vertebrate circoviruses and plant geminiviruses but with a different location for the stem-loop structure involved in rolling circle DNA replication. The replicase genes of these viruses were most closely related to those of the much smaller (approximately 1 kb) plant nanovirus circular DNA chromosomes. Because the viruses have characteristics of both animal and plant viruses, we named them chimpanzee stool-associated circular viruses (ChiSCV). Further metagenomic studies of animal samples will greatly increase our knowledge of viral diversity and evolution.

FLNA, a new partner gene fused to MLL in a patient with acute myelomonoblastic leukaemia

<i>FLNA</i>, a new partner gene fused to <i>MLL</i> in a patient with acute myelomonoblastic leukaemia

Rapid detection of ERG11 gene mutations in clinical Candida albicans isolates with reduced susceptibility to fluconazole by rolling circle amplification and DNA sequencing

BackgroundAmino acid substitutions in the target enzyme Erg11p of azole antifungals contribute to clinically-relevant azole resistance in Candida albicans. A simple molecular method for rapid detection of ERG11 gene mutations would be an advantage as a screening tool to identify potentially-resistant strains and to track their movement. To complement DNA sequencing, we developed a padlock probe and rolling circle amplification (RCA)-based method to detect a series of mutations in the C. albicans ERG11 gene using "reference" azole-resistant isolates with known mutations. The method was then used to estimate the frequency of ERG11 mutations and their type in 25 Australian clinical C. albicans isolates with reduced susceptibility to fluconazole and in 23 fluconazole-susceptible isolates. RCA results were compared DNA sequencing.ResultsThe RCA assay correctly identified all ERG11 mutations in eight "reference" C. albicans isolates. When applied to 48 test strains, the RCA method showed 100% agreement with DNA sequencing where an ERG11 mutation-specific probe was used. Of 20 different missense mutations detected by sequencing in 24 of 25 (96%) isolates with reduced fluconazole susceptibility, 16 were detected by RCA. Five missense mutations were detected by both methods in 18 of 23 (78%) fluconazole-susceptible strains. DNA sequencing revealed that mutations in non-susceptible isolates were all due to homozygous nucleotide changes. With the exception of the mutations leading to amino acid substitution E266D, those in fluconazole-susceptible strains were heterozygous. Amino acid substitutions common to both sets of isolates were D116E, E266D, K128T, V437I and V488I. Substitutions unique to isolates with reduced fluconazole susceptibility were G464 S (n = 4 isolates), G448E (n = 3), G307S (n = 3), K143R (n = 3) and Y123H, S405F and R467K (each n = 1). DNA sequencing revealed a novel substitution, G450V, in one isolate.ConclusionThe sensitive RCA assay described here is a simple, robust and rapid (2 h) method for the detection of ERG11 polymorphisms. It showed excellent concordance with ERG11 sequencing and is a potentially valuable tool to track the emergence and spread of azole-resistant C. albicans and to study the epidemiology of ERG11 mutations. The RCA method is applicable to the study of azole resistance in other fungi.

A Visual Demonstration of Plasmid DNA Preparation from Bacteria

[ILLUSTRATION OMITTED] Unprecedented advances in unraveling mysteries of life have occurred as a result of the molecular biology revolution. Some major achievements of this revolution include new methods for the production of therapeutic compounds, insights into mechanisms of gene function and regulation, and the complete sequencing of the genomes of numerous organisms including humans. A critical foundation of this revolution is the ability to use the bacterium Escherichia coli (E. coli) as a factory to produce large amounts of virtually any DNA of interest by inserting it into a small, extrachromosomal circle of DNA called a plasmid. The ease and reproducibility of plasmid DNA isolation from bacteria permits numerous labs to conduct molecular biology experiments and greatly accelerates progress in understanding complex biological processes. High school biology and introductory undergraduate biology or molecular biology classes frequently include labs in which students either isolate or manipulate plasmid DNA. Such labs often consist of a cookbook lab protocol that contains little, if any, information as to the purpose of the various steps involved in isolating the plasmid DNA. A major problem with this approach is that it leaves the procedure as a mysterious black box for most students. To assist in illustrating the roles of the different steps of the protocol, a model bacterial cell can be constructed with readily available materials and used in a highly interactive presentation that visually demonstrates the purpose of each step. This demonstration can be used as a stand-alone lesson when introducing recombinant DNA or it can be used as part of an introduction to a wet-lab experiment (a simple, rapid, low-cost, and highly reliable protocol is presented in the Appendix). The demonstration and the discussion related to it provide numerous opportunities to stress concepts included in the National Science Education Standards. * Developing Student Interest Why should I care? seems to be a common question in the minds of many students in introductory courses. Depending on the time available, the level of the students or their interests, a number of approaches can be used to help capture students' attention and engage them in class discussion. In high school freshman biology classes, we found that a means of introducing recombinant DNA technology and the role it can play in students' lives is to use an example of cloning a specific gene. Because of the prevalence of diabetes, a useful example is the cloning of the human insulin gene and the production of human insulin for therapeutic use in bacteria (Ladisch & Kohlmann, 1992). This example permits student involvement by questioning them about what they know about diabetes, the role of insulin in the disease, the health effects of this disease, or other areas the students or instructor want to develop. Since many students know someone with diabetes (in many cases they know a classmate who has diabetes or one of them may even suffer from the disease), this example provides an opportunity to get students to actively participate in discussion, regardless of their academic abilities. An alternative introduction is to discuss consequences of sequencing the entire three billion base pairs of DNA of the human genome (http://www.ornl.gov/sci/techresources/Human_ Genome/home.shtml). The current and potential uses of this technology in forensic science, health care, and business decisions are all areas that may be discussed depending on what focus the instructor wants to develop. Many students who are not normally interested in biological sciences become interested through discussions regarding possible uses of personal genetic information. Students are often surprised to learn that their ability to obtain health or life insurance or their employment may be affected by the DNA sequences they have inherited (http://www.ornl.gov/sci/ techresources/Human_Genome/elsi/elsi. …

Small polydispersed circular DNA contains strains of mobile genetic elements and occurs more frequently in permanent cell lines of malignant tumors than in normal lymphocytes

Small polydispersed circular DNA (spcDNA) belongs to the extrachromosomal pool of DNA and is composed of heterogeneous DNA circles. Whether spcDNA has a special function is currently unclear but their occurrence was suggested to be linked to genetic instability. In this study we investigated as to whether human lymphocytes from healthy volunteers also harbour spcDNA and whether spcDNA is present in all permanent cell lines from human normal and malignant tissues. Moreover, we were interested to see whether spcDNA contains sequences of mobile genetic elements. Our results show that spcDNA is present in all samples investigated yet the amount is lower in normal lymphocytes when compared to cancer cell lines (5.4 vs. 17.8%). Alu sequences were present in 12/16 cancer cell lines whereas LINE-1 (L1) sequences were present in 15 of them. Six tumor cell lines also contained telomeric sequences. In contrast to that, spcDNA of normal lymphocytes contains Alu and L1 sequences only in 3/16 cases and no telomeric sequences at all. Our findings suggest a direct dependency of the amount of Alu and L1 sequences on that of spcDNA. Beside these repetitive sequences, sequencing of spcDNA revealed in most cases chromosomal sequences of almost all chromosomes without an increased frequency of single regions. We suggest that the whole spcDNA including retrotranspositional elements and telomeric sequences may play a role for chromosomal rearrangements and genomic instability.

Hybrid Lentivirus-transposon Vectors With a Random Integration Profile in Human Cells

Gene delivery by human immunodeficiency virus type 1 (HIV-1)-based lentiviral vectors (LVs) is efficient, but genomic integration of the viral DNA is strongly biased toward transcriptionally active loci resulting in an increased risk of insertional mutagenesis in gene therapy protocols. Nonviral Sleeping Beauty (SB) transposon vectors have a significantly safer insertion profile, but efficient delivery into relevant cell/tissue types is a limitation. In an attempt to combine the favorable features of the two vector systems we established a novel hybrid vector technology based on SB transposase-mediated insertion of lentiviral DNA circles generated during transduction of target cells with integrase (IN)-defective LVs (IDLVs). By construction of a lentivirus-transposon hybrid vector allowing transposition exclusively from circular viral DNA substrates, we demonstrate that SB transposase added in trans directs efficient transposon mobilization from DNA circles in vector-transduced cells. Both transfected plasmid DNA and transduced IDLVs can serve as the source of active transposase. Most important, we demonstrate that the SB transposase overrides the natural lentiviral integration pathway and directs vector integration less frequently toward transcriptional units, resulting in a random genomic integration profile. The novel hybrid vector system combines the attractive features of efficient gene delivery by viral transduction and a safer genomic integration profile by DNA transposition.

Decreased T‐cell receptor excision DNA circles in peripheral blood mononuclear cells among benzene‐exposed workers

Benzene is a volatile aromatic hydrocarbon solvent which is widely used in many industries. The chronic exposure of humans to benzene in the workplace has been associated with blood disorders, as well as toxicity in lymphopoiesis, including aplastic anaemia and leukaemia. However, the mechanisms of benzene-induced haematotoxicity and leukaemogenesis remain unclear. In this study, we investigated the level of T-cell receptor excision DNA circles (TRECs) in peripheral blood mononuclear cells (PBMCs) in benzene-exposed workers. This would therefore be considered as a potential marker for estimates of thymic output and an evaluation of the content of naïve T-cells. It is hoped that the data will bring a comprehensive understanding on the influence of benzene exposure in the host T-cell immune function. Quantitative detection of TRECs in DNA of PBMCs from benzene-exposed workers was preformed by real-time polymerase chain reaction using the TaqMan technique. The benzene-exposed workers were divided into four groups, and 27 normal individuals were served as controls. The result indicated that the TRECs levels of all benzene-exposed groups were significantly decreased as compared with those of controls. In conclusion, the recent thymic output function and the T-cell immune function were apparently impaired in workers after benzene exposure.

Single-molecule Study of Site-specific DNA Recombination by γδ Resolvase

γδ resolvase is a serine recombinase coded by γδ transposon, which catalyzes DNA recombination between two res sites (114 bp) on a negatively supercoiled circular DNA, resulting in two catenated DNA circles. Each res site contains 3 different resolvase binding sites - site I, II and III, and each binds to a resolvase dimer. We have developed a single-DNA based system whereby synapsis and recombination should lead to torsional relaxation of a single supercoiled DNA. DNA relaxation catalyzed by γδ resolvase occurs at much higher rate on DNA substrate containing 2 res sites than those containing 1 or 0 res site. Furthermore, reactions on a 2-res-site substrate show a characteristic ∼200 nm relaxation consistent with the +4 ΔLk observed to be associated with the recombination reaction in bulk experiments. We also have observed topoisomerase activity of γδ resolvase on the non-specific (0 res) DNA substrate.