Twenty years of t-loops: A case study for the importance of collaboration in molecular biology.

Collaborative studies open doors to breakthroughs otherwise unattainable by any one laboratory alone. Here we describe the initial collaboration between the Griffith and de Lange laboratories that led to thinking about the telomere as a DNA template for homologous recombination, the proposal of telomere looping, and the first electron micrographs of t-loops. This was followed by collaborations that revealed t-loops across eukaryotic phyla. The Griffith and Tomáska/Nosek collaboration revealed circular telomeric DNA (t-circles) derived from the linear mitochondrial chromosomes of nonconventional yeast, which spurred discovery of t-circles in ALT-positive human cells. Collaborative work between the Griffith and McEachern labs demonstrated t-loops and t-circles in a series of yeast species. The de Lange and Zhuang laboratories then applied super-resolution light microscopy to demonstrate a genetic role for TRF2 in loop formation. Recent work from the Griffith laboratory linked telomere transcription with t-loop formation, providing a new model of the t-loop junction. A recent collaboration between the Cesare and Gaus laboratories utilized super-resolution light microscopy to provide details about t-loops as protective elements, followed by the Boulton and Cesare laboratories showing how cell cycle regulation of TRF2 and RTEL enables t-loop opening and reformation to promote telomere replication. Twenty years after the discovery of t-loops, we reflect on the collective history of their research as a case study in collaborative molecular biology.

Derepression of co-silenced tumor suppressor genes by nanoparticle-loaded circular ssDNA reduces tumor malignancy.

The co-silencing of multiple tumor suppressor genes can lead to escalated malignancy in cancer cells. Given the limited efficacy of anticancer therapies targeting single tumor suppressor genes, we developed small circular single-stranded DNA (CSSD) that can up-regulate the expression of co-silenced tumor suppressor genes by sequestering microRNAs (miRNAs) that negatively regulate these genes. We found that cancer patients with low tumor expression of the tumor suppressor genes KLF17, CDH1, and LASS2 had shortened survival times. The up-regulation of these genes upon transfection of artificial CSSD-9 inhibited tumor proliferation and metastasis and promoted apoptosis in vitro as well as in ex vivo and patient-derived xenograft models. In addition, CSSD is more stable and effective than current miRNA inhibitors, and transfecting CSSDs via nanoparticles substantially improved delivery efficiency. The use of a single CSSD can promote the inhibition of multiple tumor suppressor genes. This study provides evidence for the possibility of using CSSDs as therapeutic miRNA inhibitors to target the co-silencing of multiple tumor suppressor genes.

Influence of DNA sequence on the structure of minicircles under torsional stress.

The sequence dependence of the conformational distribution of DNA under various levels of torsional stress is an important unsolved problem. Combining theory and coarse-grained simulations shows that the DNA sequence and a structural correlation due to topology constraints of a circle are the main factors that dictate the 3D structure of a 336 bp DNA minicircle under torsional stress. We found that DNA minicircle topoisomers can have multiple bend locations under high torsional stress and that the positions of these sharp bends are determined by the sequence, and by a positive mechanical correlation along the sequence. We showed that simulations and theory are able to provide sequence-specific information about individual DNA minicircles observed by cryo-electron tomography (cryo-ET). We provided a sequence-specific cryo-ET tomogram fitting of DNA minicircles, registering the sequence within the geometric features. Our results indicate that the conformational distribution of minicircles under torsional stress can be designed, which has important implications for using minicircle DNA for gene therapy.

Spatial confinement induces hairpins in nicked circular DNA.

In living cells, DNA is highly confined in space with the help of condensing agents, DNA binding proteins and high levels of supercoiling. Due to challenges associated with experimentally studying DNA under confinement, little is known about the impact of spatial confinement on the local structure of the DNA. Here, we have used well characterized slits of different sizes to collect high resolution atomic force microscopy images of confined circular DNA with the aim of assessing the impact of the spatial confinement on global and local conformational properties of DNA. Our findings, supported by numerical simulations, indicate that confinement imposes a large mechanical stress on the DNA as evidenced by a pronounced anisotropy and tangent-tangent correlation function with respect to non-constrained DNA. For the strongest confinement we observed nanometer sized hairpins and interwound structures associated with the nicked sites in the DNA sequence. Based on these findings, we propose that spatial DNA confinement in vivo can promote the formation of localized defects at mechanically weak sites that could be co-opted for biological regulatory functions.

Effect of DNA bending on transcriptional interference in the systems of closely spaced convergent promoters.

BACKGROUND: Over the past years there are increasing evidences that the interplay between two molecules of RNA polymerases, initiating transcription from promoters, oriented in opposite (convergent) directions, can serve as a regulatory factor of gene expression. The data concerning the molecular mechanisms of this so-called transcriptional interference (TI) are not well understood. METHODS: The interaction of RNA polymerase with circular DNA templates, containing the convergent promoters, was investigated in a series of in vitro transcription assays and atomic force microscopy (AFM). RESULTS: In this work, to study the mechanisms of transcription interference a series of plasmids with oppositely oriented closely spaced artificial promoters, recognized by Escherichia coli RNA polymerase, was constructed. The constructs differ in promoter structure and distance between the transcription start sites. We have demonstrated that the transcripts ratio (RNA-R/RNA-L) and morphology of convergent open promoter complexes (OPC) are highly dependent on the interpromoter distance. CONCLUSIONS: The obtained results allowed us to suggest the novel model of TI, which assumes the DNA bending upon binding of RNA polymerase with promoters and explains the phenomenon of complete inactivation of weaker promoter by the stronger one. GENERAL SIGNIFICANCE: The results show that the conformational transitions in DNA helix, associated with DNA bending upon binding of RNA polymerase with promoters, play crucial role in OPC formation in the systems with convergent promoters.

Iron(II) supramolecular helicates condense plasmid DNA and inhibit vital DNA-related enzymatic activities.

The dinuclear iron(II) supramolecular helicates [Fe2 L3 ]Cl4 (L=C25 H20 N4 ) bind to DNA through noncovalent (i.e., hydrogen-bonding, electrostatic) interactions and exhibit antimicrobial and anticancer effects. In this study, we show that the helicates condense plasmid DNA with a much higher potency than conventional DNA-condensing agents. Notably, molecules of DNA in the presence of the M enantiomer of [Fe2 L3 ]Cl4 do not form intermolecular aggregates typically formed by other condensing agents, such as spermidine or spermine. The helicates inhibit the activity of several DNA-processing enzymes, such as RNA polymerase, DNA topoisomerase I, deoxyribonuclease I, and site-specific restriction endonucleases. However, the results also indicate that the DNA condensation induced by the helicates does not play a crucial role in these inhibition reactions. The mechanisms for the inhibitory effects of [Fe2 L3 ]Cl4 helicates on DNA-related enzymatic activities have been proposed.

Time-dependent bending rigidity and helical twist of DNA by rearrangement of bound HU protein.

HU is a protein that plays a role in various bacterial processes including compaction, transcription and replication of the genome. Here, we use atomic force microscopy to study the effect of HU on the stiffness and supercoiling of double-stranded DNA. First, we measured the persistence length, height profile, contour length and bending angle distribution of the DNA-HU complex after different incubation times of HU with linear DNA. We found that the persistence and contour length depend on the incubation time. At high concentrations of HU, DNA molecules first become stiff with a larger value of the persistence length. The persistence length then decreases over time and the molecules regain the flexibility of bare DNA after ∼2 h. Concurrently, the contour length shows a slight increase. Second, we measured the change in topology of closed circular relaxed DNA following binding of HU. Here, we observed that HU induces supercoiling over a similar time span as the measured change in persistence length. Our observations can be rationalized in terms of the formation of a nucleoprotein filament followed by a structural rearrangement of the bound HU on DNA. The rearrangement results in a change in topology, an increase in bending flexibility and an increase in contour length through a decrease in helical pitch of the duplex.

Strong bending of the DNA double helix.

During the past decade, the issue of strong bending of the double helix has attracted a lot of attention. Here, we overview the major experimental and theoretical developments in the field sorting out reliably established facts from speculations and unsubstantiated claims. Theoretical analysis shows that sharp bends or kinks have to facilitate strong bending of the double helix. It remains to be determined what is the critical curvature of DNA that prompts the appearance of the kinks. Different experimental and computational approaches to the problem are analyzed. We conclude that there is no reliable evidence that any anomalous behavior of the double helix happens when DNA fragments in the range of 100 bp are circularized without torsional stress. The anomaly starts at the fragment length of about 70 bp when sharp bends or kinks emerge in essentially every molecule. Experimental data and theoretical analysis suggest that kinks may represent openings of isolated base pairs, which had been experimentally detected in linear DNA molecules. The calculation suggests that although the probability of these openings in unstressed DNA is close to 10(-5), it increases sharply in small DNA circles reaching 1 open bp per circle of 70 bp.

Chain relaxation dynamics of DNA adsorbing at a solid-liquid interface.

We have used scanning force microscopy (SFM) to elucidate the dynamic behavior of open (torsionally unconstrained) circular and long linear DNA molecules during the relaxation process following adsorption onto mica. We find that bending stress and excluded volume effects drive the conformational equilibration via segmental out-of-plane dynamics.

Influence of Mg²âº, Ni²âº, and Cu²âº on DNA assembly on HOPG surfaces: atomic force microscopy study.

Adsorption of circular DNA onto bare highly oriented pyrolytic graphite (HOPG) surfaces by the addition of Mg²âº, Ni²âº, and Cu²âº has been investigated by atomic force microscopy (AFM). AFM results revealed that the topography and height of DNA on HOPG surface by the addition of different metal ions are quite different. After the addition of Mg²âº for incubation, DNA molecules tend to form many loops on HOPG surfaces, which are derived from the crossover of intramolecular and intermolecular chains. After the addition of Ni²âº, DNA molecules can form network on HOPG surfaces, and the density of DNA network was significantly increased with increasing DNA concentration. Consequently, dense DNA network can be obtained by using relatively low concentration of DNA and Ni²âº. As for the addition of Cu²âº, angular DNA loops composed of flat chains were observed. The observed flat DNA chains with an average height of 0.52 nm can be ascribed to Cu²âº insert into the site between bases and phosphate group of DNA inducing denaturation of DNA molecules. This study is very helpful for understanding the interactions of metal ions and DNA molecules, and for constructing various DNA structures on the carbonaceous surfaces.

Denaturation of circular DNA: supercoil mechanism.

The denaturation transition which takes place in circular DNA is analyzed by extending the Poland-Scheraga (PS) model to include the winding degrees of freedom. We consider the case of a homopolymer whereby the winding number of the double-stranded helix, released by a loop denaturation, is absorbed by supercoils. We find that as in the case of linear DNA, the order of the transition is determined by the loop exponent c. However the first-order transition displayed by the PS model for c>2 in linear DNA is replaced by a continuous transition with arbitrarily high order as c approaches 2, while the second-order transition found in the linear case in the regime 1

Cooperative kinking at distant sites in mechanically stressed DNA.

In cells, DNA is routinely subjected to significant levels of bending and twisting. In some cases, such as under physiological levels of supercoiling, DNA can be so highly strained, that it transitions into non-canonical structural conformations that are capable of relieving mechanical stress within the template. DNA minicircles offer a robust model system to study stress-induced DNA structures. Using DNA minicircles on the order of 100 bp in size, we have been able to control the bending and torsional stresses within a looped DNA construct. Through a combination of cryo-EM image reconstructions, Bal31 sensitivity assays and Brownian dynamics simulations, we have been able to analyze the effects of biologically relevant underwinding-induced kinks in DNA on the overall shape of DNA minicircles. Our results indicate that strongly underwound DNA minicircles, which mimic the physical behavior of small regulatory DNA loops, minimize their free energy by undergoing sequential, cooperative kinking at two sites that are located about 180° apart along the periphery of the minicircle. This novel form of structural cooperativity in DNA demonstrates that bending strain can localize hyperflexible kinks within the DNA template, which in turn reduces the energetic cost to tightly loop DNA.

Atomic force microscopy studies on circular DNA structural changes by vincristine and aspirin.

In this chapter, we have presented materials and methods to study the interaction between DNA and small molecule drugs by AFM. The detailed AFM imaging of the circular DNA after incubation with -various concentrations of vincristine and aspirin have been demonstrated. The immobilization of DNA fragments on mica surface as well as the force between tip and sample plays an important role for successful imaging of DNA-drug complexes. How to quantitatively describe the conformations and structures of circular DNA molecules and their changes is also introduced. Our work indicates that the AFM is a powerful tool in studying the interaction between DNA and small molecules.

Bending the rules of transcriptional repression: tightly looped DNA directly represses T7 RNA polymerase.

From supercoiled DNA to the tight loops of DNA formed by some gene repressors, DNA in cells is often highly bent. Despite evidence that transcription by RNA polymerase (RNAP) is affected in systems where DNA is deformed significantly, the mechanistic details underlying the relationship between polymerase function and mechanically stressed DNA remain unclear. Seeking to gain additional insight into the regulatory consequences of highly bent DNA, we hypothesize that tightly looping DNA is alone sufficient to repress transcription. To test this hypothesis, we have developed an assay to quantify transcription elongation by bacteriophage T7 RNAP on small, circular DNA templates approximately 100 bp in size. From these highly bent transcription templates, we observe that the elongation velocity and processivity can be repressed by at least two orders of magnitude. Further, we show that minicircle templates sustaining variable levels of twist yield only moderate differences in repression efficiency. We therefore conclude that the bending mechanics within the minicircle templates dominate the observed repression. Our results support a model in which RNAP function is highly dependent on the bending mechanics of DNA and are suggestive of a direct, regulatory role played by the template itself in regulatory systems where DNA is known to be highly bent.

Telomeric circles are abundant in the stn1-M1 mutant that maintains its telomeres through recombination.

Some human cancers maintain their telomeres using the alternative lengthening of telomeres (ALT) mechanism; a process thought to involve recombination. Different types of recombinational telomere elongation pathways have been identified in yeasts. In senescing yeast telomerase deletion (ter1-Delta) mutants with very short telomeres, it has been hypothesized that copying a tiny telomeric circle (t-circle) by a rolling circle mechanism is the key event in telomere elongation. In other cases more closely resembling ALT cells, such as the stn1-M1 mutant of Kluyveromyces lactis, the telomeres appear to be continuously unstable and routinely reach very large sizes. By employing two-dimensional gel electrophoresis and electron microscopy, we show that stn1-M1 cells contain abundant double stranded t-circles ranging from approximately 100 to 30,000 bp in size. We also observed small single-stranded t-circles, specifically composed of the G-rich telomeric strand and tailed circles resembling rolling circle replication intermediates. The t-circles most likely arose from recombination events that also resulted in telomere truncations. The findings strengthen the possibility that t-circles contribute to telomere maintenance in stn1-M1 and ALT cells.

An SOS inhibitor that binds to free RecA protein: the PsiB protein.

The process of bacterial conjugation involves the transfer of a conjugative plasmid as a single strand. The potentially deleterious SOS response, which is normally triggered by the appearance of single-stranded DNA, is suppressed in the recipient cell by a conjugative plasmid system centered on the product of the psiB gene. The F plasmid PsiB protein inhibits all activities of the RecA protein, including DNA binding, DNA strand exchange, and LexA protein cleavage. The proteins known to negatively regulate recombinases, such as RecA or Rad51, generally work at the level of dismantling the nucleoprotein filament. However, PsiB binds to RecA protein that is free in solution. The RecA-PsiB complex impedes formation of RecA nucleoprotein filaments on DNA.

Self-assembly of aptamer-circular DNA nanostructures for controlled biocatalysis.

Two kinds of circular DNA components are generated by the hybridization of short nucleic acids with the 3' and 5' ends of single-stranded DNA chains. The circular DNA components include, each, complementary domains for the anticocaine aptamer subunits, and sequence-specific domains for the auxiliary hybridization of programmed nucleic acid-functionalized proteins. The circular DNA components are self-assembled, in the presence of cocaine, into DNA nanowires (micrometer-long nanowires exhibiting heights of ca. 1.6-3.0 nm). Nucleic acids functionalized with glucose oxidase (GOx) and horseradish peroxidase (HRP) are hybridized with the circular DNA components to yield nanostructures consisting of HRP and GOx on the DNA scaffold. A biocatalytic cascade, where the GOx-catalyzed oxidization of glucose by O(2) yields H(2)O(2), and the resulting H(2)O(2) oxidizes 2,2'-azino-bis[3-ethylbenzthiazoline-6-sulfonic acid] (ABTS(2-)), in the presence of HRP, is activated by the system. The biocatalyzed oxidization of ABTS(2-) on the DNA scaffold is 6-fold enhanced as compared to a nonbridged homogeneous system of the two biocatalysts. The enhanced biocatalytic cascade on the DNA scaffold is attributed to high local concentrations of the reactive components in the vicinity of biocatalysts.

Bending modes of DNA directly addressed by cryo-electron microscopy of DNA minicircles.

We use cryo-electron microscopy (cryo-EM) to study the 3D shapes of 94-bp-long DNA minicircles and address the question of whether cyclization of such short DNA molecules necessitates the formation of sharp, localized kinks in DNA or whether the necessary bending can be redistributed and accomplished within the limits of the elastic, standard model of DNA flexibility. By comparing the shapes of covalently closed, nicked and gapped DNA minicircles, we conclude that 94-bp-long covalently closed and nicked DNA minicircles do not show sharp kinks while gapped DNA molecules, containing very flexible single-stranded regions, do show sharp kinks. We corroborate the results of cryo-EM studies by using Bal31 nuclease to probe for the existence of kinks in 94-bp-long minicircles.

The replication of plastid minicircles involves rolling circle intermediates.

Plastid genomes of peridinin-containing dinoflagellates are unique in that its genes are found on multiple circular DNA molecules known as 'minicircles' of approximately 2-3 kb in size, carrying from one to three genes. The non-coding regions (NCRs) of these minicircles share a conserved core region (250-500 bp) that are AT-rich and have several inverted or direct repeats. Southern blot analysis using an NCR probe, after resolving a dinoflagellate whole DNA extract in pulsed-field gel electrophoresis (PFGE), revealed additional positive bands (APBs) of 6-8 kb in size. APBs preferentially diminished from cells treated with the DNA-replication inhibitor aphidicolin, when compared with 2-3 kb minicircles, implicating they are not large minicircles. The APBs are also exonuclease III-sensitive, implicating the presence of linear DNA. These properties and the migration pattern of the APBs in a 2D-gel electrophoresis were in agreement with a rolling circle type of replication, rather than the bubble-forming type. Atomic force microscopy of 6-8 kb DNA separated by PFGE revealed DNA intermediates with rolling circle shapes. Accumulating data thus supports the involvement of rolling circle intermediates in the replication of the minicircles.

A comparative analysis of Dmc1 and Rad51 nucleoprotein filaments.

The eukaryotic RecA homologs Rad51 and Dmc1 are essential for strand exchange between homologous chromosomes during meiosis. All members of the RecA family of recombinases polymerize on DNA to form helical nucleoprotein filaments, which is the active form of the protein. Here we compare the filament structures of the Rad51 and Dmc1 proteins from both human and budding yeast. Previous studies of Dmc1 filaments suggested that they might be structurally distinct from filaments of other members of the RecA family, including Rad51. The data presented here indicate that Rad51 and Dmc1 filaments are essentially identical with respect to several structural parameters, including persistence length, helical pitch, filament diameter, DNA base pairs per helical turn and helical handedness. These data, together with previous studies demonstrating similar in vitro recombinase activity for Dmc1 and Rad51, support the view that differences in the meiotic function of Rad51 and Dmc1 are more likely to result from the influence of distinct sets of accessory proteins than from intrinsic differences in filament structure.