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PubMed1996

Induction of circles of heterogeneous sizes in carcinogen-treated cells: two-dimensional gel analysis of circular DNA molecules.

Extrachromosomal circular DNA molecules are associated with genomic instability, and circles containing inverted repeats were suggested to be the early amplification products. Here we present for the first time the use of neutral-neutral two-dimensional (2D) gel electrophoresis as a technique for the identification, isolation, and characterization of heterogeneous populations of circular molecules. Using this technique, we demonstrated that in N-methyl-N'-nitro-N-nitrosoguanidine-treated simian virus 40-transformed Chinese hamster cells (CO60 cells), the viral sequences are amplified as circular molecules of various sizes. The supercoiled circular fraction was isolated and was shown to contain molecules with inverted repeats. 2D gel analysis of extrachromosomal DNA from CHO cells revealed circular molecules containing highly repetitive DNA which are similar in size to the simian virus 40-amplified molecules. Moreover, enhancement of the amount of circular DNA was observed upon N-methyl-N'-nitro-N-nitrosoguanidine treatment of CHO cells. The implications of these findings regarding the processes of gene amplification and genomic instability and the possible use of the 2D gel technique to study these phenomena are discussed.

Animals↗

PubMed1995

Presence of a chloroplast DNA sequence in an autonomous circular DNA molecule in cultured rice cells (Oryza sativa L).

Sequence analysis of twelve DNA fragments, which had previously been found to be extensively amplified in suspension-cultured rice cells, revealed that two of them, isolated on plasmids designated pE10 and pE11, have sequences identical to distinct regions of chloroplast DNA (ct-DNA). Both sequences are part of an extrachromosomal circular DNA molecule (ECD). The molecular structure of the ECD was investigated by a combination of restriction analysis, standard and pulsed-field gel electrophoresis, hybridization with ct-DNA probes and amplification by the polymerase chain reaction in the presence of oligonucleotide primers homologous to selected regions of rice ct-DNA. The results showed that a continuous and unrearranged stretch of ct-DNA from the long single-copy region, of at least 28 kbp in length, is present in the ECD. It was estimated that the number of copies of the ECD in cultured cells was almost equivalent to that of ct-DNA molecules in rice leaves, while the ratio of ECD to ct-DNA molecules in the cultured cells was approximately 200:1.

Cells, Cultured↗

PubMed1986

Human Sau3A repeated DNA is enriched in small polydisperse circular DNA from normal lymphocytes.

Representatives of the Sau3A family of short human repeated sequences [Meneveri et al., J. Mol. Biol. 186 (1985) 483-489] have been isolated from the small polydisperse circular DNA (spcDNA) of peripheral human lymphocytes. The prototype repeat is a 72-bp element which is at least partially tandemly repeated in spcDNA and human genomic DNA. In comparison with three major families of human repeated DNA, the Sau3A repeats are enriched in spcDNA. The function of spcDNA in normal and transformed eukaryotic cells is not understood and most studies have attempted to resolve this problem by molecular analysis of circular DNA isolated from cells in culture [see Rush and Misra, Plasmid 14 (1985) 177-191 for references]. We have studied the spcDNA present in normal uncultured human lymphocytes and present data pointing to the selective accumulation of the Sau3A family of repeated DNA within this population. The sequences of twelve of these repeats, the consensus sequence for this family and the sequence of a genomic repeat, are presented.

Base Sequence↗

PubMed1976

Small circular DNA in Drosophila melanogaster.

Covalently closed small circular DNA isolated from Drosophila melanogaster is described. The small circular DNA is found in blastema stage eggs and in Schneider's cell culture line 2 and a cloned subline of line 2. It is heterogeneous in size, although the size distributions and mean sizes differ for each source. The small circular DNA from Schneider's line 2 cells ranges from 0.09-7.3 mum, with a mean contour length of 1.1 mum. This DNA has a buoyant density of 1.703 g/cc and appears to be present predominantly in the nuclear fraction of detergent-disrupted cells. the restriction enzyme EcoRl cleaves approximately 40% of the small circular DNA with a bias toward the larger size classes. Both logarithmic and stationary phase cells contain approximately 3-40 average sized small circular DNA molecules per cell, representing a maximum of 0.03% of the total cellular DNA. Exposure to cycloheximide or puromycin for 14 hr results in a 30 fold increase in the number of small circles per cell, but reduces the mean length of the circular DNA to 0.3 mum. The drug-amplified DNA has a buoyant density in the range of 1.698-1.703 g/cc. No amplification was seen in cells treated with either inhibitor for 3.5 hr. Ethidium bromide, cytosine arabinoside, beta-ecdysone, and insulin all had no significant effect on the amount per cell of either small circular DNA or mitochondrial DNA.

Animals↗

PubMed1986

New, small circular DNA in transfected mammalian cells.

Circular DNA isolated by the Hirt procedure from transfected mammalian cells was examined by electron microscopy. Typically, the number of small (1- to 5-kilobase) DNA circles increased about fivefold even though DNA of larger size classes (5 to 15 kilobases) has been transferred. In one case, where extensive rearrangement of the transferred DNA was observed, the rearrangement products were cloned and analyzed. In most cases, however, no rearrangement could be detected, but the amount of small circular DNA was still increased. This effect was seen with two transfection procedures (erythrocyte ghost fusion and calcium phosphate precipitation) and with various combinations of transfecting DNA and recipient cell type. The origin of the new small circular DNA is discussed.

Animals↗

PubMed1983

Mechanism of stimulation by a specific protein factor of de novo DNA synthesis by mouse DNA replicase with fd phage single stranded circular DNA.

Mouse DNA replicase is a functional multienzyme complex consisting of DNA polymerase and DNA primase. The DNA and initiator RNA syntheses by DNA replicase with single stranded DNA as template are stimulated by a stimulating factor (T. Yagura, T. Kozu and T. Seno, 1982, J. Biochem. (Tokyo).91, 607-618). The action mechanism of the stimulating factor on this novel DNA synthesis with fd phage single stranded circular DNA as template was studied. The stimulating factor directly stimulated initiator RNA synthesis but did not change the length of either initiator RNA (8 to 10 nucleotides long) or the product DNA (300 to 1,000 nucleotides long). Kinetic studies and analysis of the products by neutral agarose gel electrophoresis show that the stimulating factor increased the affinity of DNA replicase for template DNA without changing the apparent Km values for deoxy- and ribonucleotide substrates. Thus, in combination with a sufficient amount of the stimulating factor, DNA replicase quantitatively converted the template DNA to the position of double-stranded circular replicative form II DNA, as shown by agarose gel electrophoresis.

Animals↗

PubMed1985

Triple rings: a new type of compact structure of circular DNA.

Complexes of circular superhelical pBR322 DNA with a synthetic tripeptide capable of beta-structure formation (dansylhydrazide trivaline) were studied at different peptide/DNA ratios by electron microscopy. It was shown on rotary-shadowed preparations that peptide binding induces intramolecular DNA condensation and compact ring-shaped particles are formed from fibres 120 A thick. The analysis of the morphology of the ring structures observed at various peptide/DNA ratios as well as contour length measurements enabled us to draw conclusions about the organization of the double-stranded DNA filaments in these structures. It was established that the fibres forming compact rings contain three double-stranded DNA segments closely associated due to DNA-peptide and peptide-peptide interactions. The mechanisms leading to the formation of the triple rings may be important in DNA condensation in vivo.

DNA, Circular↗

PubMed2006

Structural perturbations induced in linear and circular DNA by the architectural protein HU from Bacillus stearothermophilus.

HU is a small DNA-binding protein of eubacteria that is believed to induce or stabilize bending of the double helix and mediate nucleoid compaction in vivo. Although HU does not bind preferentially to specific DNA sequences, it is known to have high affinity for DNA sites containing structural anomalies, such as unpaired or mismatched bases, nicks, and four-way junctions. We have employed Raman spectroscopy to further investigate the structural basis of HU-DNA recognition in solution. Experiments were carried out on the homodimeric HU protein of Bacillus stearothermophilus (HUBst) and a 222-bp DNA fragment, which was isolated in linear (DNA(L222)) and circular (DNA(C222)) forms. In the absence of bound HUBst the Raman signatures of DNA(L222) and DNA(C222) are nearly superimposable, indicating that circularization produces no substantial change in the local B-DNA conformation. Conversely, the Raman signatures of DNA(L222) and DNA(C222) are perturbed significantly and specifically by HUBst binding. The HUBst-induced perturbations are markedly greater for the circularized DNA target. These results support an opportunistic molecular mechanism, in which HU binding is facilitated by intrinsic nonlinearity or flexibility in the DNA target. We propose that DNA segments which are bent or predisposed toward bending provide the high-affinity sites for HU attachment and nucleoid condensation. This model is consistent with the wide range of DNA bending angles reported in crystal structures of HU-DNA complexes.

Bacterial Proteins↗

PubMed1966

Physical and topological properties of circular DNA.

Several types of circular DNA molecules are now known. These are classified as single-stranded rings, covalently closed duplex rings, and weakly bonded duplex rings containing an interruption in one or both strands. Single rings are exemplified by the viral DNA from phiX174 bacteriophage. Duplex rings appear to exist in a twisted configuration in neutral salt solutions at room temperature. Examples of such molecules are the DNA's from the papova group of tumor viruses and certain intracellular forms of phiX and lambda-DNA. These DNA's have several common properties which derive from the topological requirement that the winding number in such molecules is invariant. They sediment abnormally rapidly in alkaline (denaturing) solvents because of the topological barrier to unwinding. For the same basic reason these DNA's are thermodynamically more stable than the strand separable DNA's in thermal and alkaline melting experiments. The introduction of one single strand scission has a profound effect on the properties of closed circular duplex DNA's. In neutral solutions a scission appears to generate a swivel in the complementary strand at a site in the helix opposite to the scission. The twists are then released and a slower sedimenting, weakly closed circular duplex is formed. Such circular duplexes exhibit normal melting behavior, and in alkali dissociate to form circular and linear single strands which sediment at different velocities. Weakly closed circular duplexes containing an interruption in each strand are formed by intramolecular cyclization of viral lambda-DNA. A third kind of weakly closed circular duplex is formed by reannealing single strands derived from circularly permuted T2 DNA. These reconstituted duplexes again contain an interruption in each strand though not necessarily regularly spaced with respect to each other.

Bacteriophages↗

PubMed1989

Infectious circular DNA of human adenovirus type 5: regeneration of viral DNA termini from molecules lacking terminal sequences.

A series of plasmids containing the entire human adenovirus genome with viral DNA termini joined 'head to tail' has been isolated. Several plasmids were able to generate infectious virus following transfection of human cells in spite of having small deletions and rearrangements at the junctions of termini. One plasmid has lost 2 bp of DNA from one end of the viral genome and 11 bp from the other end yet produced viruses with complete wild-type sequences at both ends of the genome. We propose a model for replication of viral DNA off circular templates in which regeneration of terminal information involves translocation of primer and polymerase during initiation of DNA replication. The model suggests a novel mechanism for extension of the 5' ends of linear DNA molecules which could be applicable to chromosomal telomeres.

Adenoviruses, Human↗

PubMed2003

Biochemical analysis of the yeast condensin Smc2/4 complex: an ATPase that promotes knotting of circular DNA.

To better understand the contributions that the structural maintenance of chromosome proteins (SMCs) make to condensin activity, we have tested a number of biochemical, biophysical, and DNA-associated attributes of the Smc2p-Smc4p pair from budding yeast. Smc2p and Smc4p form a stable heterodimer, the "Smc2/4 complex," which upon analysis by sedimentation equilibrium appears to reversibly self-associate to form heterotetramers. Individually, neither Smc2p nor Smc4p hydrolyzes ATP; however, ATPase activity is recovered by equal molar mixing of both purified proteins. Hydrolysis activity is unaffected by the presence of DNA. Smc2/4 binds both linearized and circular plasmids, and the binding appears to be independent of adenylate nucleotide. High mole ratios of Smc2/4 to plasmid promote a geometric change in circular DNA that can be trapped as knots by type II topoisomerases but not as supercoils by a type I topoisomerase. Binding titration analyses reveal that two Smc2/4-DNA-bound states exist, one disrupted by and one resistant to salt challenge. Competition-displacement experiments show that Smc2/4-DNA-bound species formed at even high protein to DNA mole ratios remain reversible. Surprisingly, only linear and supercoiled DNA, not nicked-circular DNA, can completely displace Smc2/4 prebound to a labeled, nicked-circular DNA. To explain this geometry-dependent competition, we present two models of DNA binding by SMCs in which two DNA duplexes are captured within the inter-coil space of an Smc2/4 heterodimer. Based on these models, we propose a DNA displacement mechanism to explain how differences in geometry could affect the competitive potential of DNA.

Adenosine Triphosphatases↗

PubMed1986

Multiple mechanisms generate extrachromosomal circular DNA in Chinese hamster ovary cells.

Seven cloned small circular DNA molecules from CHO cells were sequenced and examined for the presence of homologies to each other and to a number of other functional sequences present in transposable elements, retroviruses, mammalian repeat sequences, and introns. The sequences of the CHO cell circular DNA molecules did not reveal common structural features that could explain their presence in the circular DNA population. A gene bank was constructed for CHO chromosomal DNA and sequences homologous to two of the seven small circular DNA molecules were isolated and sequenced. The nucleotide sequences present at the junction of circular and chromosomal DNA suggest that a recombination process involving homologous pairing may have been involved in the generation of one, but not the other, of the two circular DNA molecules.

Animals↗

PubMed2006

Quantification of hepatitis B virus covalently closed circular DNA in patients with hepatocellular carcinoma.

BACKGROUND/AIMS: This study aimed to measure the intrahepatic total hepatitis B virus (HBV) DNA and covalently closed circular DNA (cccDNA) levels in tumor and non-tumor tissues in hepatocellular carcinoma (HCC) patients. METHODS: Intrahepatic total HBV DNA and cccDNA were measured in 25 HCC patients (21 hepatitis B surface antigen [HBsAg]-positive and 4 HBsAg-negative) by the Invader assay. RESULTS: A low level of intrahepatic HBV DNA was detectable in all HBsAg-negative patients. For HBsAg-positive patients, the intrahepatic total HBV DNA levels in the tumor and non-tumor tissues were comparable (P=0.903). However, the tumor tissues had significantly higher levels of cccDNA (0.35 vs. 0.16 copies/cell, P=0.030) and higher proportion of intrahepatic HBV DNA in the form of cccDNA (100% vs. 84%, P=0.004) than the non-tumor tissues. Seventeen out of 21 (81%) tumor tissues had intrahepatic HBV DNA solely in cccDNA form. Analysis of HBV mRNA expression indicated that HBV replication appeared to be lower in the tumor tissues than the non-tumor tissues. CONCLUSIONS: Compared to the non-tumor tissues, the levels of HBV replication in the tumor tissues appeared to be lower, and cccDNA was the predominant form of HBV DNA in the tumor tissues.

Adult↗

PubMed1987

Absence in monocotyledonous plants of the diffusible plant factors inducing T-DNA circularization and vir gene expression in Agrobacterium.

T-DNA circularization is one of the molecular events specifically induced in agrobacterial cells upon their infection of dicotyledonous plant cells. We developed a seedling co-cultivation procedure to determine whether or not monocotyledonous plants have the ability to induce T-DNA circularization and vir gene expression. Co-cultivation of Agrobacterium tumefaciens with seedlings of dicotyledonous plants showed that the circularization event takes place efficiently. The exudates and extracts of the seedlings also effectively induced T-DNA circularization and vir gene expression, indicating that dicotyledonous seedlings contain diffusible factors capable of inducing these molecular events. In contrast, neither T-DNA circularization nor vir gene expression was detectable when Agrobacterium was incubated with seedlings of monocotyledonous plants. Supplementing with acetosyringone, a known inducer of vir gene expression and T-DNA circularization, resulted in the induction of circularization during co-cultivation with moncotyledonous seedlings. These results indicate that the seedlings of monocotyledonous plants have no detectable amounts of diffusible inducers, unlike dicotyledonous seedlings. Therefore, it is unlikely that the vir genes are expressed in Agrobacterium inoculated in monocotyledonous plants. This may be one of the blocks in tumorigenesis of monocotyledonous plant by Agrobacterium.

DNA, Bacterial↗

PubMed1999

Topology effect for DNA structure of cisplatin: topological transformation of cisplatin-closed circular DNA adducts by DNA topoisomerase I.

The reaction of cis-Pt(NH3)2Cl2(cis-DDP)-closed circular DNA adducts with DNA topoisomerse I(topo I) were studied by electron microscopy. We identified unique topoisomers such as a singly-linked catenane (2(1)2), trefoil (3(1), and dimetric catenane (2(1)2), etc., by analysis with electron micrographs. These unique recombination products resulted from cis-DDP-intra-twisting looped DNA adducts by DNA topo I, and the products could be explained a new mechanism based on an odd-even number rule. Our results suggest a new model on the working mechanisms for DNA topology of cis-DDP which enhances the recombination of DNA. Based on our results, we propose the topological idea that the yields of a mini closed circular DNA and pseudo trefoil DNA, etc., can be expected by reaction of cis-DDP-DNA-histone complexes with DNA topo I in the body.

Antineoplastic Agents↗

PubMed1978

Organization of mammalian chromosomal DNA: supercoiled and folded circular DNA subunits from interphase cell nuclei.

Chinese hamster cells (line V79/4) in the G2 phase of the mitotic cycle were lysed onto neutral sucrose gradients and the released chromosomal DNA was characterized according to its size and shape by sedimentation velocity studies. Using the intercalating agent, ethidium bromide, in the gradients and the induction of DNA single- and double-strand breaks by irradiation, the DNA was proved to be released into the gradient in supercoiled circular subunits whose homogenous size corresponds to 2.8 . 10(9) Dalton. Supercoiling of these DNA subunits was found to be confined to smaller regions sized on the average about 9 . 10(7) Dalton and maintained by folding the DNA into loops. The average superhelix density was determined to be--0.09 turns per 10 base pairs of DNA. The functional aspects of the experimental findings are discussed in terms of replicative and transcriptional units.

Cell Cycle↗

PubMed1989

Isolation of an IgH gene circular DNA clone from human bone marrow.

Circular DNA was obtained from human bone marrow. Then a phage library was prepared and screened by use of two probes of the IgH gene; 5'-DHQ52, containing the 5' flanking region of DHQ52, and JH4.3, containing the sequence from JH3 to the 3' flanking region of JH6. One clone, HBMC-1, that was DHQ52+JH4.3- was obtained. HBMC-1 had the germline IgH region upstream of JH1 and the 3' flanking region of DXP1. A recombination signal sequence flanking the 5' side of the JH1 segment was attached to the recombination signal sequence flanking the 3' side of DXP1 forming a head-to-head structure of two 7mers with 10 nucleotides in-between. HBMC-1 is thus considered to be a circular DNA deleted as a consequence of DXP1-JH1 joining of the IgH gene.

Base Sequence↗

PubMed1995

DNA enzymology above 100 degrees C. Topoisomerase V unlinks circular DNA at 80-122 degrees C.

The widespread application of polymerase chain reaction and related techniques in biology and medicine has led to a heightened interest in thermophilic enzymes of DNA metabolism. Some of these enzymes are stable for hours at 100 degrees C, but no enzymatic activity on duplex DNA at temperatures above 100 degrees C has so far been demonstrated. Recently, we isolated topoisomerase V from the hyperthermophile Methanopyrus kandleri, which grows up to 110 degrees C. This novel enzyme is similar to eukaryotic topoisomerase I and acts on duplex DNA regions. We now show that topoisomerase V catalyzes the unlinking of double-stranded circular DNA at temperatures up to 122 degrees C. In this in vitro system, maximal DNA unlinking occurs at 108 degrees C and corresponds to complementary strands being linked at most once. These results further imply that in the presence of sufficient positive supercoiling DNA can exist as a double helix even at 122 degrees C.

DNA Topoisomerases, Type I↗