Branched oligonucleotides containing bicyclic nucleotides as branching points and DNA or LNA as triplex forming branch

Various Y-shaped branched oligonucleotides containing a 2'-0,3'-C-ethylene linked or 2'-0,4'-C-methylene linked bicyclic nucleotide as branching point were synthesized on an automated DNA synthesizer. Thermal denaturation experiments at 260 and 284 nm showed increased thermal stabilities of complexes formed between these Y-shaped oligonucleotides and complementary DNA compared with those formed with the corresponding linear reference. The most significant effect was observed when LNA (locked nucleic acid) monomers were used in the triplex forming branch.     

Torsional control of double-stranded DNA branch migration

DNA branched junctions are analogues of Holliday junction recombination intermediates. Partially mobile junctions contain a limited amount of homology flanking the branch point. A partially mobile DNA branched junction has been incorporated into a synthetic double-stranded circular DNA molecule. The junction is flanked by four homologous nucleotide pairs, so that there are five possible locations for the branch point. Two opposite arms of the branched junction are joined to form the circular molecule, which contains 262 nucleotides to the base of the junction. This molecule represents a system whereby torque applied to the circular molecule can have an impact on the junction, by relocating its branch point. Ligation of the molecule produces two topoisomers; about 87% of the product is a relaxed molecule, and the rest is a molecule with one positive supercoil. The position of the branch point is assayed by cleaving the molecule with endonuclease VII. We find that the major site of the branch point in the relaxed topoisomer is at the maximally extruded position in the relaxed molecule. Upon the addition of ethidium, the major site of the branch point migrates to the minimally extruded position. © 1998 John Wiley & Sons, Inc. Biopoly 45: 69–83, 1998     

RECQ1 possesses DNA branch migration activity

RecQ helicases are essential for the maintenance of genome stability. Five members of the RecQ family have been found in humans, including RECQ1, RECQ5, BLM, WRN, and RECQ4; the last three are associated with human diseases. At this time, only BLM and WRN helicases have been extensively characterized, and the information on the other RecQ helicases has only started to emerge. Our current paper is focused on the biochemical properties of human RECQ1 helicase. Recent cellular studies have shown that RECQ1 may participate in DNA repair and homologous recombination, but the exact mechanisms of how RECQ1 performs its cellular functions remain largely unknown. Whereas RECQ1 possesses poor helicase activity, we found here that the enzyme efficiently promotes DNA branch migration. Further analysis revealed that RECQ1 catalyzes unidirectional three-stranded branch migration with a 3' --> 5' polarity. We show that this RECQ1 activity is instrumental in specific disruption of joint molecules (D-loops) formed by a 5' single-stranded DNA invading strand, which may represent dead end intermediates of homologous recombination in vivo. The newly found enzymatic properties of the RECQ1 helicase may have important implications for the function of RECQ1 in maintenance of genomic stability.     

DNA branch nuclease activity of vaccinia A22 resolvase

DNA replication, recombination, and repair can result in formation of diverse branched DNA structures. Many large DNA viruses are known to encode DNA branch nucleases, but several of the expected activities have not previously been found among poxvirus enzymes. Vaccinia encodes an enzyme, A22 resolvase, which is known to be active on four-stranded DNA junctions (Holliday junctions) or Holliday junction-like structures containing three of the four strands. Here we report that A22 resolvase in fact has a much wider substrate specificity than previously appreciated. A22 resolvase cleaves Y-junctions, single-stranded DNA flaps, transitions from double strands to unpaired single strands ("splayed duplexes"), and DNA bulges in vitro. We also report site-directed mutagenesis studies of candidate active site residues. The results identify the likely active site and support a model in which a single active site is responsible for cleavage on Holliday junctions and splayed duplexes. Lastly, we describe possible roles for the A22 resolvase DNA-branch nuclease activity in DNA replication and repair.     

Discrete and continuous mathematical models of DNA branch migration

DNA junctions, known as Holliday junctions, are intermediates in genetic recombination between DNAs. In this structure, two double-stranded DNA helices with similar sequence are joined at a branch point. The branch point can move along these helices when strands with the same sequence are exchanged. Such branch migration is modeled as a random walk. First, we model this process discretely, such that the motion of the branch is represented as transfer between discrete compartments. This is useful in analysing the results of DNA branch migration on junction comprised of synthetic oligonucleotides. The limit in which larger numbers of smaller steps go to continuous motion of the branch is also considered. We show that the behavior of the continuous system is very similar to that of the discrete system when there are more than just a few compartments. Thus, even branch migration on oligonucleotides can be viewed as a continuous process. One consequence of this is that a step size must be assumed when determining rate constants of branch migration.We compare migration where forward and backward movements of the branch are equally probable to biased migration where one direction is favored over the other. In the latter case larger differences between the discrete and continuous cases are predicted, but the differences are still small relative to the experimental error associated with experiments to measure branch migration in oligonucleotides.     

Important points in virus research using recombinant DNA technology

Cartagena Protocol on Biosafety to the Convention on Biological Diversity seeks to protect biological diversity from potential risks posed by living modified organisms (LMOs) resulting from modern biotechnology. This protocol was ratified in Japan after establishing domestic law and regulations for the protocol. In the domestic law, use of LMOs is classified into type 1 use (use without containment measures) and type 2 use (use with containment measures). According to the domestic law, most of experiments using recombinant viruses are required for the approval of the Minister. In this article, we will explain Cartagena Protocol and the Japanese domestic low and indicate an example of application form for the approval of the Minister.     

Accumulation of DNA growing points in caffeine-treated human lymphoblastoid cells

DNA bifilarly substituted with bromodeoxyuridine (DNA_HH) can be obtained after incubation of human lymphoblastoid cells with heavy analog for short periods. The DNA_HH originates from DNA growing points and is derived by branch migration in vitro during or after cell lysis. Treatment of the cells with caffeine resulted in an increase in both the relative and absolute amount of DNA_HH as compared to non-treated cultures synthesizing equivalent amounts of DNA. In conjunction with the smaller size of the nascent DNA synthesized in the presence of caffeine, the accumulation of DNA_HH likely indicates an accumulation of growing forks, possibly resulting from the decreased rate of chain elongation and the initiation of replication at additional sites of origin. The usefulness of measurement of DNA_HH as a probe to estimate the number of replicating forks, i.e. the number of active replicons, is discussed.     

Direct evidence for spontaneous branch migration in antiparallel DNA Holliday junctions

The Holliday junction is a central intermediate in genetic recombination. It contains four strands of DNA that are paired into four double helical arms flanking a branch point. In naturally occurring Holliday junctions, the sequence flanking the branch point contains 2-fold (homologous) symmetry. As a consequence of this symmetry, the junction can undergo a conformational isomerization known as branch migration, which relocates the site of branching. In the absence of proteins and in the presence of Mg(2+), the four arms are known to stack in pairs, forming two helical domains whose orientations are antiparallel. Nevertheless, the mechanistic models proposed for branch migration are all predicated on a parallel alignment of helical domains. Here, we have used antiparallel DNA double crossover molecules to demonstrate that branch migration can occur in antiparallel Holliday junctions. We have constructed a DNA double crossover molecule with three crossover points. Two adjacent branch points in this molecule are flanked by symmetric sequences. The symmetric crossover points are held immobile by the third crossover point, which is flanked by asymmetric sequences. Restriction of the helices that connect the immobile junction to the symmetric junctions releases this constraint. The restricted molecule undergoes branch migration, even though it is constrained to an antiparallel conformation.     

DnaB drives DNA branch migration and dislodges proteins while encircling two DNA strands

DnaB is a ring-shaped, hexameric helicase that unwinds the E. coli DNA replication fork while encircling one DNA strand. This report demonstrates that DnaB can also encircle both DNA strands and then actively translocate along the duplex. With two strands positioned inside its central channel, DnaB translocates with sufficient force to displace proteins tightly bound to DNA with no resultant DNA unwinding. Thus, DnaB may clear proteins from chromosomal DNA. Furthermore, while encircling two DNA strands, DnaB can drive branch migration of a synthetic Holliday junction with heterologous duplex arms, suggesting that DnaB may be directly involved in DNA recombination in vivo. DnaB binds to just one DNA strand during branch migration. T7 phage gp4 protein also drives DNA branch migration, suggesting this activity generalizes to other ring-shaped helicases.     

DNA recombination: holliday junctions dynamics and branch migration

Holliday junctions are critical intermediates for homologous, site-specific recombination, DNA repair, and replication. A wealth of structural information is available for immobile four-way junctions, but the controversy on the mechanism of branch migration of Holliday junctions remains unsolved. Two models for the mechanism of branch migration were suggested. According to the early model of Alberts-Meselson-Sigal (Sigal, N., and Alberts, B. (1972) J. Mol. Biol. 71, 789-793 and Meselson, M. (1972) J. Mol. Biol. 71, 795-798), exchanging DNA strands around the junction remain parallel during branch migration. Kinetic studies of branch migration (Panyutin, I. G., and Hsieh, P. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 2021-2025) suggest an alternative model in which the junction adopts an extended conformation. We tested these models using a Holliday junction undergoing branch migration and time-lapse atomic force microscopy, an imaging technique capable of imaging DNA dynamics. The single molecule atomic force microscopy experiments performed in the presence and in the absence of divalent cations show that mobile Holliday junctions adopt an unfolded conformation during branch migration that is retained despite a broad range of motion in the arms of the junction. This conformation of the junction remains unchanged until strand separation. The data obtained support the model for branch migration having the extended conformation of the Holliday junction.     

Three-dimensional structural views of branch migration and resolution in DNA homologous recombination

The processing of the Holliday junction by various proteins is a major event in DNA homologous recombination and is crucial to the maintenance of genome stability and biological diversity. The proteins RuvA, RuvB and RuvC play central roles in the late stage of recombination in prokaryotes. Recent atomic views of these proteins, including protein-protein and protein-junction DNA complexes, provide new insights into branch migration mechanisms: RuvA is likely to be responsible for base-pair rearrangements, whereas RuvB, classified as a member of the AAA(+) family, functions as a pump to pull DNA duplex arms without segmental unwinding. The mechanism of junction resolution by RuvC in the RuvABC resolvasome remains to be elucidated.     

Four exons of the serotonin receptor 4 gene are associated with multiple distant branch points

Splicing of vertebrate introns involves recognition of three consensus elements at the 3′ end. The branch point (BP) and polypyrimidine tract (PPT) are usually located within 40 nucleotides (nt) of the 3′ splice site (3′ ss), AG, but can be much more distant. A characteristic of the region between distant BPs (dBPs) and the 3′ ss is the absence of intervening AG dinucleotides, leading to its designation as the “AG exclusion zone” (AGEZ). The human HTR4 gene, which encodes serotonin receptor 4 and has been associated with schizophrenia, bipolar disease, and gastrointestinal disorders, has four exons with extensive AGEZs. We have mapped the BPs for HTR4 exons 3, 4, 5, and g generated by in vitro splicing, and validated them by mutagenesis in exon-trapping vectors. All exons used dBPs up to 273 nt upstream of the exon. Strikingly, exons 4 and 5 used combinations of both distant and conventionally located BPs, suggesting that successful splicing of these exons can occur by distinct pathways. Our results emphasize the importance for single nucleotide polymorphism resequencing projects to take account of potential dBPs, as the extended AGEZs are vulnerable to mutations that could affect splicing itself or regulation of alternative splicing.     

Reassociation rate limited displacement of DNA strands by branch migration.

Large branched DNA structures are constructed by two-step reassociation of separated complementary strands from restriction fragments of different lengths. The displacement of DNA strands initially annealed to longer complementary DNA sequences, a process mediated by branch migration, is very rapid and has thus far been followed only under conditions which are second order, DNA reassociation rate limiting. The average lifetime of branched DNA leading to displacement of 1.6 Kb strands is estimated to be less than 10 seconds under conditions of DNA reassociation, Tm-25 degrees C. Several DNA-binding drugs, including intercalating dyes, have been tested to determine their influence, if any, on the kinetics of DNA strand displacements by branch migration. Only actinomycin D was found to have significant effect under the conditions we have described. The kinetics of the strand displacement in the presence of low concentrations of actinomycin D remain second order and slower rate of strand displacement must be attributed to decreased rate of reassociation of DNA strands to form the branched intermediates. Consideration is given to the potential manipulation of DNA structures at site-directed branches and the limitations due to rapid strand displacements. The feasibility of constructing sufficiently large branched DNA regions to approach first order, branch migration rate limiting kinetics is also discussed.     

Vemurafenib-resistant BRAF selects alternative branch points different from its wild-type BRAF in intron 8 for RNA splicing

One mechanism of resistance of the melanoma-associated BRAF kinase to its small molecule inhibitor vemurafenib is by point mutations in its intron 8 resulting in exons 4–8 skipping. In this report, we carried out in vitro BRAF RNA splicing assays and lariat RT-PCR to map the intron 8 branch points in wild-type and BRAF mutants. We identify multiple branch points (BP) in intron 8 of both wild-type (wt) and vemurafenib-resistant BRAF RNA. In wt BRAF, BPs are located at -29A, -28A and -26A, whereas in a vemurafenib-resistant BRAF splicing mutant, BPs map to -22A, -18A and -15A, proximal to the intron 8 3′ splice site. This finding of a distal-to-proximal shift of the branch point sequence in BRAF splicing in response to point-mutations in intron 8 provides insight into the regulation of BRAF alternative splicing upon vemurafenib resistance.     

Septin 6 regulates the cytoarchitecture of neurons through localization at dendritic branch points and bases of protrusions

Septins, a conserved family of GTP-binding proteins with a conserved role in cytokinesis, are present in eukaryotes ranging from yeast to mammals. Septins are also highly expressed in neurons, which are post-mitotic cells. Septin6 (SEPT6) forms SEPT2/6/7 complexes in vivo. In this study, we produced a very specific SEPT6 antibody. Immunocytochemisty (ICC) of dissociated hippocampal cultures revealed that SEPT6 was highly expressed in neurons. Developmentally, the expression of SEPT6 was very low until stage 3 (axonal outgrowth). Significant expression of SEPT6 began at stage 4 (outgrowth of dendrites). At this stage, SEPT6 clusters were positioned at the branch points of developing dendrites. In maturing and mature neurons (stage 5), SEPT6 clusters were positioned at the base of filopodia and spines, and pre-synaptic boutons. Detergent extraction experiments also indicated that SEPT6 is not a post-synaptic density (PSD) protein. Throughout morphologic development of neurons, SEPT6 always formed tiny rings (external diameter, ∼0.5 μm), which appear to be clusters at low magnification. When a Sept6 RNAi vector was introduced at the early developmental stage (DIV 2), a significant reduction in dendritic length and branch number was evident. Taken together, our results indicate that SEPT6 begins to be expressed at the stage of dendritic outgrowth and regulates the cytoarchitecture.     

Two-dimensional maps of short-term albumin uptake by the immature and mature rabbit aortic wall around branch points

In children, aortic lipid deposition develops in triangular regions of the wall downstream of branch points, whilst in adults these regions are particularly free of disease. Comparable age-related patterns occur in rabbit aortas. They may be explained by patterns of wall permeability to circulating macromolecules: along the longitudinal midline through branches, permeability is greater downstream than upstream in immature rabbits, but is greater upstream at later ages. Here we have mapped permeability in detail around such branches, not just along the midline. Short-term uptake of rhodamine-labeled albumin, measured using digital imaging fluorescence microscopy of serial sections, was greatest in an approximately triangular region downstream of immature branches, but in mature animals it was greater upstream, particularly away from the midline, and in streaks to the side of branches. Hence the maps are consistent with earlier permeability data and closely resemble the patterns of disease.     

Principles of branch formation and branch patterning in Hydrozoa

The freshwater polyp Hydra produces buds which separate from the parent. Other Hydrozoa produce branches which remain connected to the parent, thus forming a colony. Some Hydrozoa grow by means of an organ that is like a shoot apical meristem. Others display a sympodial type of growth. In this article, I propose that these different types of branches are organized by a common pattern-forming system. This system has self-organizing properties. It causes branch tip formation and is kept active in the tip when the tip finally differentiates into a hypostome of a polyp. The system does not cause structure formation directly but rather, determines a tissue property called positional value, in such a way that a gradient of values forms in the tissue of the bud or branch. The local value determines the local morphodynamic processes, including differentiation of the hypostome (highest positional value), tentacles and basal disc and of the exoskeleton pattern along the shoot. A high positional value favors the onset of a new self-organizing process and by lateral inhibition, such a process prevents the initiation of a further process in its surroundings. Small quantitative differences in the range of the signals involved determine whether a bud or a branch forms and whether monopodial and sympodial growth follows.