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PubMed2006

Stable triple-stranded DNA formation and its application to the SNP detection.

We have found that a short stretch (30mer or larger) of triple-stranded DNA structure formed at the terminus (or very near) of linear DNA molecules is unusually stable, withstanding heat treatment at as high as 95 degrees C. The stable triple-stranded structure is formed only when deoxyoligonucleotides are complementary to the strand terminating with 5'-phosphate and not to the strand terminating with 3'-OH. Presence of a single mismatched base in the complementary deoxyoligonucleotides drastically reduces the stability. We show that these unique properties of the terminal triple-stranded structure can be applied to the detection of single nucleotide polymorphisms in genomes without DNA dissociation and/or hybridization.

Base Pairing↗

PubMed1986

(A-T)n tracts embedded in random sequence DNA--formation of a structure which is chemically reactive and torsionally deformable.

Alternating d(A-T)n sequences which are contiguous with DNA of effectively random sequence have an abnormal conformation in linear DNA molecules. These regions are strongly reactive towards chemical modification by osmium tetroxide, and are preferentially cleaved by micrococcal nuclease. Both the chemical modification and the enzymic cutting occur uniformly through the alternating tract, and there is no evidence for enzyme or chemical sensitivity in the interfaces between the tract and DNA of normal conformation. These reactivities have a requirement for an alternating sequence. In addition to chemical reactivity, alternating (A-T)n sequences exhibit anomalously small twist changes on cruciform formation, suggesting that the pre-extruded DNA is underwound. We propose that the alternating sequences adopt an altered conformation which is subject to easy torsional deformation.

Base Sequence↗

PubMed2002

The terminal repeats and latency-associated nuclear antigen of herpesvirus saimiri are essential for episomal persistence of the viral genome.

The simian herpesvirus saimiri (HVS) induces malignant T cell lymphomas and is closely related to Kaposi's sarcoma-associated herpesvirus (KSHV or HHV-8). Both belong to the gamma-2 herpesvirus subgroup. The viral genome of HVS consists of a unique region (L-DNA) that contains all of the viral genes flanked by non-coding terminal repeats (H-DNA). Here we describe the cloning of a 113 kb restriction fragment containing the L-DNA of an oncogenic HVS strain in an F' replicon-based E. coli vector. Cloned DNA was infectious and the ends of the progeny viral genome consisted of amplified tandem alternating repeats of vector and a single H-DNA unit. T cells infected with these viruses contained the linear DNA typically found a few weeks after infection, but were unable to form episomal circular viral DNA, which is the latent form of the viral genome. Recombinant viruses with reconstructed H-DNA were generated and T cells infected with these rescued viruses contained high copy numbers of episomal DNA. Plasmids expressing the latency-associated nuclear antigen (LANA) and containing various numbers of H-DNA repeats stably replicated as episomes, but constructs containing three repeat units produced the highest copy numbers. These data show that intact and multiple terminal repeats are essential components for episomal replication in latently infected T cells. Moreover, LANA and terminal repeats are sufficient for stable plasmid persistence. Cloned HVS can also be utilized for mutagenesis of HVS and for the expression of foreign genes through efficient manipulation of plasmids in E. coli.

Animals↗

PubMed1999

Double-strand-break repair recombination in Escherichia coli: physical evidence for a DNA replication mechanism in vivo.

DNA double-strand-break repair (DSBR) is, in many organisms, accomplished by homologous recombination. In Escherichia coli DSBR was thought to result from breakage and reunion of parental DNA molecules, assisted by known endonucleases, the Holliday junction resolvases. Under special circumstances, for example, SOS induction, recombination forks were proposed to initiate replication. We provide physical evidence that this is a major alternative mechanism in which replication copies information from one chromosome to another generating recombinant chromosomes in normal cells in vivo. This alternative mechanism can occur independently of known Holliday junction cleaving proteins, requires DNA polymerase III, and produces recombined DNA molecules that carry newly replicated DNA. The replicational mechanism underlies about half the recombination of linear DNA in E. coli; the other half occurs by breakage and reunion, which we show requires resolvases, and is replication-independent. The data also indicate that accumulation of recombination intermediates promotes replication dramatically.

Bacterial Proteins↗

PubMed2004

The push-pull mechanism of bacteriophage Ø29 DNA injection.

The mechanism of bacteriophage DNA injection is poorly understood, often considered a simple process, driven merely by the packing pressure inside the capsid. In contrast to the well-established DNA packaging mechanism of Bacillus subtilis phage Ø29, that involves a molecular motor formed by the connector and a viral ATPase, nothing is known about its DNA injection into the cell. We have studied this process measuring DNA binding of p6, a viral genome organization protein. The linear DNA penetrates with a right-left polarity, in a two-step process. In the first step approximately 65% of the genome is pushed into the cell most probably by the pressure built inside the viral capsid. Thus, synthesis of viral proteins from the right early operon is allowed. This step is controlled, probably by bacterial protein(s) that slow down DNA entry. In the second step at least one of the viral early proteins, p17, participates in the molecular machinery that pulls the remaining DNA inside the cell. Both steps are energy-dependent, as treatment of cells with azide overrides the whole mechanism, leading to a deregulated, passive entry of DNA.

Bacillus Phages↗

PubMed2006

Base excision repair in mammalian cells.

A rapid, convenient and safe in vitro assay system for base excision repair is described. Whole cell extracts are prepared by detergent-based cell lysis and provide a vigorous activity of AP site repair. A circular DNA substrate is used for detection of both DNA polymerase beta-dependent and proliferating cell nuclear antigen (PCNA)-dependent pathways. Repaired and unrepaired DNA substrates are separated by agarose gel electrophoresis as a linear DNA molecule and a nicked circular molecule, respectively, and detected by staining with SYBR Green I. This assay system does not require radioactive substrates or nucleotides, and provides a sensitivity in which 10 ng of a DNA substrate per reaction is sufficient for quantitative repair analysis.

Animals↗

PubMed1999

DNase II in spermatozoa of the loach misgurnus fossilis L

A deoxyribonuclease (DNase) which is active at acid pH in the absence of bivalent cations was found in loach spermatozoa. The enzyme was purified by ion-exchange chromatography and partially characterized. The DNase has optimal activity at pH 5.5 and its molecular weight is about 30 kD; its substrate is covalently closed circular duplex DNA, and its product is the corresponding unit-length linear DNA. The DNase is inhibited by MgCl2 and activated by EDTA. Thus, this endoDNase found in loach spermatozoa can be classified as a DNase II. The biological role of this DNase II is discussed.

Journal Article↗

PubMed1996

Direct visualization of dispersed 11q13 chromosomal translocations in mantle cell lymphoma by multicolor DNA fiber fluorescence in situ hybridization.

Several hematologic malignancies are associated with specific chromosomal translocations. Because of the dispersed distribution, chromosomal breakpoints may be difficult to detect using molecular techniques. We present a new application of a recently developed method, DNA fiber fluorescence in situ hybridization (fiber FISH), which allows direct visualization and mapping of chromosomal breakpoints. We tested this method for detection of the t(11;14)(q13;q32) translocation in mantle cell lymphoma. In DNA fiber FISH, a series of fluorochrome-labeled DNA probes covering several hundreds of kilobasepairs is hybridized to linear DNA molecules (or fibers) prepared from frozen tissue or intact cells. By using alternate fluorescent colors, a potential breakpoint region is stained in a color barcode pattern. Breaks in this region will split the barcode in two complementary parts, from which the breakpoint position can be derived. We used a 250-kb barcode covering the BCL-1 locus to detect 11q13 breakpoints in 20 well-characterized mantle cell lymphomas. A t(11;14) was shown by cohybridization of these probes with probes for the Ig heavy chain locus at 14q32. In 18 of 20 mantle cell lymphomas, a breakpoint within the 11q13/BCL-1 barcode was shown by the presence of multiple, complementary translocation products. Fusion of 11q13 and 14q32 sequences on single fibers indicating t(11;14)(q13;q32) was found in all 18 breakpoint-positive mantle cell lymphomas. In one additional case, fusion of an intact 11q13 barcode with 14q32 sequences indicated a breakpoint 100 kb centromeric of the major translocation cluster of BCL-1. Within the 120-kb region of BCL-1, breakpoints were widely scattered. This explains why, so far, a BCL-1 breakpoint had been detected by Southern blot analysis in only 10 of 19 cases. DNA fiber FISH analysis showed a t(11;14) in 95% of mantle cell lymphoma. The results indicate that DNA fiber FISH is a rapid, simple, and equally powerful method for detection of clustered and dispersed translocation breakpoints.

Chromosome Aberrations↗

PubMed1996

Fiber-FISH: experiences and a refined protocol.

One of the most time-consuming steps in positional cloning is the physical mapping of probes from the critical chromosomal region and the assembly of a genomic contig of large insert probes. New high-resolution Fiber-FISH techniques have significantly facilitated this tedious task by enabling rapid direct visualization of the order, degree of overlap and gap sizes of adjacent large insert clones. We have developed a method, where agarose-embedded DNA (PFGE block) is used as a source for preparing linearized DNA targets on microscope slides. This modification of the fiber-FISH technique has been successfully used in physical mapping in the 1-300 kb range as well as for detecting genomic rearrangements. Here, we present a refined protocol of our original technique. The application of this technique to agarose embedded yeast cells is also demonstrated. Finally, critical steps and trouble shooting of the method are addressed.

Chromosome Mapping↗

PubMed1992

Cleavage by restriction enzymes of DNA modified with the antitumour drug cis-diamminedichloroplatinum(II).

The effect of binding of an antitumour drug cis-diamminedichloroplatinum(II) (cis-[Pt(NH3)2Cl2]) to DNA on cutting effectiveness of BamHI, EcoRI, and SalI restriction endonucleases was quantitatively determined. The platinum complex inhibits the cleavage of plasmid pHC624 DNA linearized by BglI restrictase. From the present results we conclude that the yield of restriction endonuclease cleavage is also lowered if the platinum complex is bound outside the recognition DNA sequence of these enzymes. We propose that the origin of platinum adducts on DNA outside the recognition sequence can decrease the yield of restriction enzyme cleavage via inducing a conformational perturbation in the recognition DNA sequence of these enzymes and also via inhibition of the linear diffusion of these enzymes on DNA.

Base Sequence↗

PubMed1988

Identification of homologous pairing and strand-exchange activity from a human tumor cell line based on Z-DNA affinity chromatography.

An enzymatic activity that catalyzes ATP-dependent homologous pairing and strand exchange of duplex linear DNA and single-stranded circular DNA has been purified several thousand-fold from a human leukemic T-lymphoblast cell line. The activity was identified after chromatography of nuclear proteins on a Z-DNA column matrix. The reaction was shown to transfer the complementary single strand from a donor duplex linear substrate to a viral circular single-stranded acceptor beginning at the 5' end and proceeding in the 3' direction (5'----3'). Products of the strand-transfer reaction were characterized by electron microscopy. A 74-kDa protein was identified as the major ATP-binding peptide in active strand transferase fractions. The protein preparation described in this report binds more strongly to Z-DNA than to B-DNA.

Adenosine Triphosphatases↗

PubMed2000

Size conversion of a linear plasmid in the relapsing fever agent Borrelia duttonii.

Borreliae have genomes composed of both linear and circular replicons. We have characterized the organization of linear DNA molecules from the Borrelia duttonii strain Ly. It contains a linear one megabase chromosome and 12 linear plasmids of 11 to 200 kb in size. A variant of the strain obtained after successive in vitro cultivation in BSKII medium had a 69 kb molecule instead of the 44 kb linear plasmid. No detectable differences in the growth rates and cellular structures were found. Southern hybridization using the vsp33 gene sequence from Borrelia hermsii as a probe showed that both plasmids (69 and 44 kb molecules) contained a similar part of the sequence. The spirochetes of the parental strain cause erythrocytes to aggregate in mice blood, but the variant did not form such aggregates and seemed to have lost its infectivity in mice. Size conversion of the linear plasmid may be associated with the host-parasite relationship in mammals.

Animals↗

PubMed1986

Eukaryotic topoisomerase II. Characterization of enzyme turnover.

While the binding of adenyl-5'-yl imidodiphosphate (App(NH)p) to Drosophila melanogaster topoisomerase II induces a double-stranded DNA passage reaction, its nonhydrolyzable beta,gamma-imidodiphosphate bond prevents enzyme turnover (Osheroff, N., Shelton, E. R., and Brutlag, D. L. (1983) J. Biol. Chem. 258, 9536-9543). Therefore, this ATP analog was used to characterize the interactions between Drosophila topoisomerase II and DNA which occur after DNA strand passage but before enzyme turnover. In the presence of App(NH)p, a stable post-strand passage topoisomerase II-nucleic acid complex is formed when circular DNA substrates are employed. Although noncovalent in nature, these complexes are resistant to increases in ionic strength and show less than 5% dissociation under salt concentrations (greater than 500 mM) that disrupt 95% of the enzyme-DNA interactions formed in the absence of App(NH)p or under a variety of other conditions that do not support DNA strand passage. These results strongly suggest that the process of enzyme turnover not only regenerates the active conformation of topoisomerase II but also confers upon the enzyme the ability to disengage from its nucleic acid product. Experiments with linear DNA molecules indicate that after strand passage has taken place, topoisomerase II may be able to travel along its DNA substrate by a linear diffusion process that is independent of enzyme turnover. Further studies demonstrate that the regeneration of the enzyme's catalytic center does not require enzyme turnover, since topoisomerase II can cleave double-stranded DNA substrates after strand passage has taken place. Finally, while the 2'-OH and 3'-OH of ATP are important for its interaction with Drosophila topoisomerase II, neither are required for turnover.

Adenosine Diphosphate↗

PubMed2004

Protelomerase uses a topoisomerase IB/Y-recombinase type mechanism to generate DNA hairpin ends.

Protelomerases are enzymes responsible for the generation of closed hairpin ends in linear DNA. It is proposed that they use a breaking-and-rejoin type mechanism to affect DNA rearrangement on specific DNA sequences. In doing so, one strand turns around and becomes the complementary strand. Using the purified enzyme from the Escherichia coli phage N15 and the Klebsiella phage phiKO2 and synthetic oligonucleotide substrates, we directly demonstrate the location where the cutting/re-ligation occurs. We identified a pair of transient staggered cleavages six base-pairs apart centered around the axis of dyad symmetry of the target site. Two molecules of the protelomerase form a pair of protein-linked DNA intermediates at each 3' end of the cleaved openings leaving a 5'-OH. Then, in a process not yet clearly defined, the partners of the two initial openings are exchanged, and the transient breaks are resealed to generate hairpin ends. The formation of 3'-covalent DNA-protein intermediates is a hallmark of the topoisomerase IB type reaction, and we have thus shown experimentally that protelomerase is a member of the tyrosine-recombinase superfamily. In addition, by introducing single nicks in the substrates as perturbation, we found that the integrity of the nucleotide chain 4 bp away from the cutting site as well as this nucleotide's complementary location on the stem if the strands were to fold into a cruciform structure are required for activity, suggesting that these locations may be important substrate-protein contacts. We determined that N15 and phiKO2 protelomerases are monomers in solution and two molecules are needed to interact with the substrate to form two closed hairpin products. The target sites of protelomerases invariably consist of inverted repeats. Comparative studies using the related target sites of different protelomerases suggest that these proteins may require both sequence-specific and structure (possibly cruciform)-specific recognition for activity.

Base Sequence↗

PubMed1997

Generation of catalytic RNAs by rolling transcription of synthetic DNA nanocircles.

Small catalytic RNAs are commonly produced either by transcription of promoter-driven linear DNA templates or by stepwise chemical synthesis on solid supports. We describe a different approach, in which very small chemically synthesized circular DNAs serve as efficient templates for generation of catalytic RNAs in vitro. The circles are 83 nucleotides in size, are single stranded, and contain no canonical RNA polymerase promoters. Despite this, T7 and Escherichia coli RNA polymerases transcribe the circles by a rolling mechanism, producing long concatemeric RNAs (approximately 7,500 nt). During the transcription reaction, the repeating RNAs self-cleave, ultimately reaching monomer length. Despite having self-complementary sequences at their substrate-binding domains, these monomeric 83-nt RNAs are shown to be catalytically active ribozymes that sequence-specifically cleave RNA targets in trans. In addition, a circular vector encoding a repeating (non-self-processing) ribozyme is described; the resulting multimeric ribozyme, targeted to a sequence in the HIV-1 genome, is also catalytically active in trans. This novel approach to the synthesis of catalytic RNAs offers a number of differences and potential advantages over current approaches to RNA synthesis.

DNA, Circular↗

PubMed2002

Human foamy virus integrase fails to catalyse the integration of a circular DNA molecule containing an LTR junction sequence.

The presence of closed circular forms of the linear DNA genome of human foamy virus (HFV) has not been established. The ability of the HFV integrase (IN) to catalyse the integration of these circular forms (termed 2 long terminal repeat (LTR) circles) was investigated, with a view to producing a novel hybrid vector. To this end, a construct was made containing, in addition to the enhanced green fluorescent protein (eGFP) marker gene, the last 27 bp of the 3' U5 LTR region of HFV fused to the first 28 bp of the 5' U3 LTR, the latter representing a 2LTR circle. Marker gene expression following transfection of both 293 and 293T cells indicated that the level of integration was not significantly increased by the HFV IN. Moreover, correctly integrated provirus-like forms of the input plasmid could not be detected by PCR. Taken together, these results show that the HFV IN is not able to integrate a circular molecule containing an LTR junction and, hence, the technique is not exploitable as a tool to produce hybrid vectors for gene therapy.

Catalysis↗

PubMed2003

Origin usage during euplotes ribosomal DNA amplification.

The macronuclear genome of the ciliate Euplotes is comprised of millions of small linear DNA molecules that have telomeres on each end. These molecules are generated during the sexual stage of the life cycle, when the new macronucleus is formed by a series of DNA processing events and multiple rounds of DNA amplification. We have used two-dimensional gels to compare the location of the replication origins used during vegetative growth and the two periods during macronuclear development when DNA amplification takes place. When we examined the pattern of ribosomal DNA (rDNA) replication intermediates, we observed almost identical Y arcs regardless of when in the Euplotes life cycle the DNA was isolated. No bubble or bubble-to-Y arcs could be detected. This indicates that replication of the macronuclear rDNA initiates at or near the telomere even when these molecules are being differentially amplified. Since replication rarely initiated from both ends of the rDNA, we examined the direction of replication fork movement to determine which end of the rDNA served as the origin. Fork movement gels indicated that replication initiated at the 5' end. As transcription also starts near the telomere at the 5' end, our findings suggest that the telomere and the promoter region cooperate to recruit Euplotes replication initiation complexes.

Animals↗

PubMed2003

[Redusome aging: commentaries].

The redusome hypothesis of aging and biological age control (Olovnikov, Biochemistry (Moscow) 2003, vol. 68, pp. 2-33; http://protein.bio.msu.su/biokhimiya/contents/v68/ToC6801.htm.) is discussed. Though the main part of telomere-related predictions (Olovnikov, 1971, 1973) have successfully been confirmed (end under-replication of linear DNA molecules; explanation why bacterial genome is circled to avoid this problem; telomerase existence in sex and cancer cells; correlation of telomera shortening with the number of cell doublings already performed by somatic cells that divide and age in vitro), I state that telomere model of cell aging should be abandoned, since a telomere-dependent signal of cellular senescence does not exist. Instead, it is postulated that so called redusomes are involved in control of biological time and aging. Redusomes are postulated nuclear organelles which are presented by small linear double helix DNA molecules of different specificities which are covered by proteins and located at special chromosomal nests. Each redusome has its own ori for replication, as well as promoter for transcription, but it has no centromere. Hence redusomes are distributing in mitoses among daughter cells only due to the behavior of chromosomes as their specific carriers. Transcripts from redusomes (both micro RNAs and so called fountain RNAs) participate in chromatin remodeling and chromosomal structural genes expression. Regular and consecutive losses of repeated genes from chronomeres (DNA of redusomes of neuroendoclinal and neurotrophic cells of a brain) are perceived by cells of brain's biochronometer as a course of biological time. Continuation of shortening of redusomal DNA molecules in the organism that has already achieved its physiological maturity is responsible both for cellular senescence and the organism aging. Telomere attrition is only a bystander process of aging, while the genuine cause of the cell and organism aging is the redusome DNA shortening.

Aging↗