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PubMed1992

Antitumor quinolones with mammalian topoisomerase II mediated DNA cleavage activity.

Ninety quinolones were evaluated to determine whether their ability to induce mammalian topoisomerase II mediated DNA cleavage in vitro correlated with their antitumor activity in vivo. Ten quinolones generated linear DNA at a yield of more than 10% of substrate supercoiled DNA in the mammalian topoisomerase II mediated DNA cleavage assay. All of these compounds showed a significant increase in life span (greater than 20%) in the murine leukemia P388 model. These antitumor quinolones have closely related structures: two halogens at C-6 and C-8; and cyclopropyl at N-1 of quinolone skeleton. In contrast, many analogues of the above quinolones, as well as new quinolones used clinically as an antibacterial drug, did not induce the cleavable complex in vitro or show antitumor activity in vivo. These findings indicate that quinolone derivatives can be a promising new class of antitumor agent targeting mammalian topoisomerase II.

Animals↗

PubMed1993

Intoplicine (RP 60475) and its derivatives, a new class of antitumor agents inhibiting both topoisomerase I and II activities.

Intoplicine (RP 60475, NSC 645008) is an antitumor derivative in the 7H-benzo[e]pyrido[4,3-b]indole series which is now being tested in clinical trials. Intoplicine strongly binds DNA (KA = 2 x 10(5) M-1) and thereby increases the length of linear DNA. These properties are consistent with DNA unwinding by intoplicine. Intoplicine was found to be a dual topoisomerase I and II inhibitor, with DNA sites of enzyme inhibition being different for these two enzymes. In this study, 22 analogues of intoplicine were evaluated for their effects on topoisomerase I- and II-mediated DNA cleavage reactions by using enzymes purified from calf thymus. Site-specific DNA cleavage mediated by topoisomerase I was observed with 7H-benzo[e]pyrido[4,3-b]indole derivatives but not with 11H-benzo[g]-pyrido[4,3-b]indole derivatives. Site-specific DNA cleavage mediated by topoisomerase II occurred with derivatives having hydroxyl groups at the 3-position on the 7H-benzo[e]pyrido[4,3-b]indole ring or at the 4-position on the 11H-benzo[g]pyrido[4,3-b]indole ring. Study of the relationships between the in vivo antitumor activity on P388 leukemia and the topoisomerase I- and/or II-mediated DNA cleavage activity revealed that the most highly active antitumor compounds possessed both topoisomerase I-and II-inhibitory properties. Compounds selectively inhibiting either topoisomerase I or II were less active. These results suggest that dual topoisomerase I and II inhibition is critical for the antitumor activity of this new series of antitumor compounds.

Animals↗

PubMed1986

Microinjected Tetrahymena rDNA ends are not recognized as telomeres in Xenopus eggs.

Telomeres are essential structures that stabilize the ends of eukaryotic chromosomes and allow complete replication of linear DNA molecules. We examined the structure and replication of telomeres by observing the fate of the linear extrachromosomal rDNA of Tetrahymena after injection into unfertilized Xenopus eggs. The rDNA replicated efficiently as a linear extrachromosomal molecule, increasing in mass 30-50-fold by 15-20 h after injection. In addition, the molecules increased in length by addition of up to several kilobases of DNA to their termini. Sequence analysis demonstrated that the added DNA bore no resemblance to known telomeres. The junction between the rDNA and added DNA was apparently random, indicating that the addition reaction did not involve a site-specific recombination or integration event. Surprisingly, Southern blot analysis showed that the added DNA did not derive from Xenopus DNA, but rather from co-purifying and therefore co-injected Tetrahymena DNA. The nonspecific ligation of random DNA fragments to the rDNA termini suggests that microinjected Tetrahymena rDNA ends are not recognized as telomeres in Xenopus eggs.

Animals↗

PubMed1994

Efficient synthesis of 32P-labeled single-stranded DNA probes using linear PCR; application of the method for analysis of strand-specific DNA repair.

We have developed a rapid method to synthesize radioactively labeled single-stranded DNA probes suitable for strand-specific analysis of single copy genes on Southern blot. Linear PCR with 10 microCi alpha 32P-dATP (3000 Ci/mmol) as the only dATP source enabled us to generate strand-specific DNA probes with high specific activity. The probes synthesized by this method have higher specific activities and the same strand specificity compared to the end-labeled single-stranded DNA probes obtained from single-stranded M13mp18/19 vectors. Application of the method for strand-specific analysis of ultraviolet-induced DNA lesions in defined DNA sequences significantly improved the hybridization signal.

Base Sequence↗

PubMed1996

Cell cycle-regulated generation of single-stranded G-rich DNA in the absence of telomerase.

Current models of telomere replication predict that due to the properties of the polymerases implicated in semiconservative replication of linear DNA, the two daughter molecules have one end that is blunt and one end with a short 3' overhang. Telomerase is thought to extend the short 3' overhang to produce long single-stranded overhangs. Recently, such overhangs, or TG1-3 tails, were shown to occur on both telomeres of replicated linear plasmids in yeast. Moreover, indirect evidence suggested that the TG1-3 tails also occurred in a yeast strain lacking telomerase. We report herein a novel in-gel hybridization technique to probe telomeres for single-stranded DNA. Using this method, it is shown directly that in yeast strains lacking the TLC1 gene encoding the yeast telomerase RNA, TG1-3 single-stranded DNA was generated on chromosomal and plasmid telomeres. The single-stranded DNA only appeared in S phase and was sensitive to digestion with a single-strand-specific exonuclease. These data demonstrate that during replication of telomeres, TG1-3 tails can be generated in a way that is independent of telomerase-mediated strand elongation. In wild-type strains, these TG1-3 tails could subsequently serve as substrates for telomerase and telomere binding proteins on all telomeres.

Base Composition↗

PubMed1995

A transformation system for the yeast Candida utilis: use of a modified endogenous ribosomal protein gene as a drug-resistant marker and ribosomal DNA as an integration target for vector DNA.

We have developed a transformation system for the yeast Candida utilis. A novel strategy was applied to construct the transformation system, since auxotrophic mutants which could be used as hosts for transformation are not available. A gene encoding the ribosomal protein L41 was cloned from C. utilis, which is sensitive to cycloheximide, and used as a marker gene conferring cycloheximide resistance after modification of its amino acid sequence. The marker gene was constructed by substitution of the proline codon at position 56 with the glutamine codon by in vitro mutagenesis, as it had been reported previously that the 56th amino acid residue of L41 is responsible for the cycloheximide sensitivity of various organisms (S. Kawai, S. Murao, M. Mochizuki, I. Shibuya, K. Yano, and M. Takagi, J. Bacteriol. 174:254-262 1992). The ribosomal DNA (i.e., DNA coding for rRNA) of C. utilis was also cloned and used as a multiple-copy target for the integration of vector DNA into the genome, which resulted in a high transformation efficiency. Transformants were obtained by electroporation with a maximum efficiency of approximately 1,400 transformants per 1 microgram of linearized DNA carrying the gene for cycloheximide resistance and part of the ribosomal DNA. No transformants were obtained with intact plasmids. Multiple copies of the linearized plasmid were integrated into the host chromosome by homologous recombination. Southern analysis of the transformants in which vector DNA was integrated at the L41 gene locus indicated that there are two copies of gene for the L41 protein per cell, suggesting that C. utilis is diploid. Transformants were obtained from a variety of C. utilis strains, indicating that this method is applicable to the transformation of other C. utilis strains, even though there is significant heterogeneity in chromosomal karyotypes among these strains.

Amino Acid Sequence↗

PubMed2000

Radiolysis of nucleosome core DNA: a modelling approach.

PURPOSE: To calculate the expected pattern of frank strand breaks (FSB) induced in the DNA of a nucleosome core particle (NCP) by the attack of radiolytic OH* radicals and to compare this with the experimental pattern of FSB induced by the in vitro irradiation of chicken NCP. MATERIALS AND METHODS: The structure of NCP was obtained from the PDB databank. Using molecular modelling, the structure of a linear DNA fragment with the central 60 bp sequence of NCP-DNA was determined. The accessibility of the sugar attack sites to OH* radicals at each nucleotide of the linear fragment or of the NCP-DNA was calculated. The probability of reaction of OH* with each sugar reactive site was calculated using a Monte-Carlo method-based stochastic model. RESULTS: The accessibility of attack sites in the NCP-DNA and the calculated probabilities of sugar attack are mainly modulated through masking by histones, and only slightly through bending. The periodicity of the calculated FSB probabilities is identical to that of the experimental FSB probabilities in chicken NCP (period 10.4 +/- 0.1 bp). CONCLUSIONS: The good agreement between the calculated and experimental results recommends the presented modelling procedure as a tool in predicting the radiosensitivity of DNA in DNA-ligand complexes of known structure.

Animals↗

PubMed1993

Two-dimensional motion of DNA bands during 120 degrees pulsed-field gel electrophoresis.

The position and velocity of a band of double-stranded, linear DNA from bacteriophage G were measured during 120 degrees pulsed-field gel electrophoresis, using a video micrometer. Both the x and y coordinates were determined simultaneously in the plane of a 1% agarose gel; x is the mean drift direction. For pulse durations T greater than the tube renewal time T*, the path traced by the band of 670 kb DNA in the xy plane was in remarkably good accord with that predicted by Southern's ratchet model. However, the measured instantaneous velocity vx showed a sharp backward spike each time the field changed direction, with amplitude about twice the mean drift velocity. This spike is not consistent with models which assume a constant curvilinear velocity of DNA in a tube, nor with the biased reptation model without fluctuations. The corresponding measurements of vy showed a sharp positive spike with amplitude more than 3 times the plateau velocity in the y direction; neither model predicted this. The sharp velocity spikes are consistent with the idea that, for T > T*, a large fraction of the DNA chains are stretched into U-shaped or herniated configurations. When the field changes direction, the arms of the U's and the hernias recoil rapidly in response to intramolecular DNA chain tension. Because the base of a U or hernia is fixed by gel fibers, the center of mass of the chain recoils backward every time the field changes direction.

Chemical Phenomena↗

PubMed1979

Torsional stress and local denaturation in supercoiled DNA.

It is shown that local denaturation can be a natural consequence of supercoiling, even in environments where base pairing of linear DNA is energetically favored. Any change in the molecular total twist from its unstressed value is partitioned between local denaturation and smooth twisting in both the native and coil regions so as to minimize the total conformational free energy involved. Threshold degrees of torsional deformation are found for the existence of stable, locally melted conformations. As these thresholds are surpassed, the number of denatured bases increase smoothly from zero. Existing experimental evidence regarding denaturation in supercoiled DNA is in good agreement with the predictions of this theory. In addition, from existing data one can estimate the partitioning of superhelicity between twisting and writhing. Possible consequences of stress-induced strand separation on the accessibility of the DNA to enzyme attack are discussed. Control of local melting by DNA topoisomerases and DNA gyrases could regulate diverse events involved in transcription, replication, recombination, and repair.

Base Sequence↗

PubMed1999

Ku-dependent nonhomologous DNA end joining in Xenopus egg extracts.

An extract from activated Xenopus eggs joins both matching and nonmatching ends of exogenous linear DNA substrates with high efficiency and fidelity (P. Pfeiffer and W. Vielmetter, Nucleic Acids Res. 16:907-924, 1988). In mammalian cells, such nonhomologous end joining (NHEJ) is known to require the Ku heterodimer, a component of DNA-dependent protein kinase. Here I investigated whether Ku is also required for the in vitro reaction in the egg extract. Immunological assays indicate that Ku is very abundant in the extract. I found that all NHEJ was inhibited by autoantibodies against Ku and that NHEJ between certain combinations of DNA ends was also decreased after immunodepletion of Ku from the extract. The formation of a joint between a DNA end with a 5'-protruding single strand (PSS) and an end with a 3'-PSS, between two ends with 3'-PSS, and between two blunt ends was most Ku dependent. On the other hand, NHEJ between two DNA ends bearing 5'-PSS was Ku independent. These results show that the Xenopus cell-free system will be useful to biochemically dissect the role of Ku in eukaryotic NHEJ.

Animals↗

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↗

PubMed1997

The effect of ionic conditions on the conformations of supercoiled DNA. II. Equilibrium catenation.

We studied the equilibrium formation of DNA catenanes to assess the conformational properties of supercoiled DNA as a function of ionic conditions and supercoiling density. Catenanes were formed by cyclizing linear DNA with long cohesive ends in the presence of supercoiled molecules. The efficiency of the catenation depends on the distance between opposing segments of DNA in the interwound superhelix. The fraction of cyclizing molecules that becomes topologically linked with the supercoiled DNA is the product of the concentration of the supercoiled DNA and a proportionality constant, B, that depends on conformations of supercoiled DNA. In parallel with these experimental studies, we calculated the values of B using Monte Carlo simulations of the equilibrium distribution of DNA conformations. There were no adjustable parameters in the calculations because all three parameters of the DNA model, bending and torsional elasticity of DNA and DNA effective diameter, specifying intersegment interactions, were known from independent studies. We found very good agreement between measured and simulated values of B for all the ionic conditions and DNA superhelix densities studied; the discrepancy was less than a factor of 2 over the 200-fold variation in B. The value of B decreases nearly exponentially with increasing superhelicity, this dependence being especially strong at low salt concentration. The dependence of B on the concentration of NaCl, MgCl(2), and spermidine can be described with good accuracy in terms of changes of the DNA effective diameter. We found no indication of superhelix collapse under any ionic conditions studied. We discuss, in light of these results, the biological importance of the effect of DNA supercoiling on the unlinking of the products of DNA replication.

Computer Simulation↗

PubMed1999

Double-strand DNA hydrolysis by dilanthanide complexes.

Although there has been progress in developing artificial hydrolytic DNA cleaving agents, none of these has been shown to carry out the double-strand hydrolysis of DNA. We demonstrate that La(III) or Ce(IV) combined with the ligand 1,3-diamino-2-hydroxypropane-N,N,N', N'-tetraacetate (HPTA) in a 2 : 1 ratio can efficiently cleave supercoiled plasmid DNA at 55 degrees C within a 3-h period. Analysis of end-labeled restriction fragments cleaved by these complexes reveals 3'- and 5'-ends consistent with a hydrolytic mechanism. Unlike for other polydentate carboxylate complexes, plasmid DNA cleavage by La(2)(HPTA) or Ce(2)(HPTA) affords a significant amount of linear DNA with a considerable fraction of the supercoiled form still remaining. This result implies that La(2)(HPTA) and Ce(2)(HPTA) can carry out double-strand cleavage of plasmid DNA. La(2)(HPTA) and Ce(2)(HPTA) represent the first metal complexes demonstrated to be capable of double-strand hydrolytic cleavage of plasmid DNA.

DNA↗

PubMed2006

A survey of expressed tRNA genes in the chromosome I of Arabidopsis using an RNA polymerase III-dependent in vitro transcription system.

Eukaryotic tRNA genes are transcribed by RNA polymerase III. These tRNA genes are generally predicted using computer programs, and 620 tRNA genes in the Arabidopsis thaliana genome are currently annotated. However, no effort has been made to assay whether these predicted tRNA genes are all expressed, because it has been difficult to assay by routine in vivo methods. We report here a large-scale tRNA expression assay of predicted Arabidopsis tRNA genes using an RNA polymerase III-dependent in vitro transcription system developed by our group. DNA fragments including an annotated tRNA gene each were amplified by PCR and the resulting linear DNA was subjected to in vitro transcription. The addition of poly(dA-dT).poly(dA-dT) enhanced activity significantly and reduced background. The 124 predicted tRNA genes present in the Arabidopsis chromosome I were examined, and transcription activity and transcript stability from individual genes were determined. These results indicated that eight annotated genes are not expressed. Based on previous reports on pseudo-tRNA genes (e.g., Beier and Beier, Mol. Gen. Genet. 1992; 233: 201-208) and the present results, we estimated that 16% or more of the annotated tRNA genes in the chromosome I are not functional.

5' Flanking Region↗

PubMed2000

Intercalating residues determine the mode of HMG1 domains A and B binding to cisplatin-modified DNA.

Cisplatin exerts its anticancer activity by forming covalent adducts with DNA. High-mobility group (HMG)-domain proteins recognize the major 1,2-intrastrand cisplatin-DNA cross-links and can mediate cisplatin cytotoxicity. The crystal structure of HMG1 domain A bound to cisplatin-modified DNA, further analyzed here, reveals intercalation of a key Phe37 residue. Other published structures of HMG domains bound to DNA, including NHP6A and HMG-D, similarly indicate amino acid side chains intercalating into linear DNA to form a bend. To delineate the importance of such side chain intercalations and further to explore the binding modes of different HMG domains toward prebent DNA structures, site-directed mutagenesis was used to generate HMG1 domain A and domain B mutants. The affinities of these mutant proteins for cisplatin-modified DNA were determined in gel electrophoresis mobility shift assays. The results indicate that intercalating residues at positions 16 or 37 can both contribute to the binding affinity. The data further reveal that the length of the loop between helices I and II is not critical for binding affinity. Footprinting analyses indicate that the position of the intercalating residue dictates the binding mode of the domain toward platinated DNA. Both congruent and offset positioning of the HMG domain with respect to the locus of the cisplatin-induced bend in the DNA were encountered. Packing interactions in the crystal structure suggest how full-length HMG1 might bind to DNA by contacting more than one duplex simultaneously. Taken together, these results demonstrate that cisplatin modification of DNA provides an energetically favorable, prebent target for HMG domains, which bind to these targets through one or more side chain and favorable hydrophobic surface interactions.

Amino Acid Sequence↗

PubMed1998

Four differently chromatin-associated maize HMG domain proteins modulate DNA structure and act as architectural elements in nucleoprotein complexes.

In contrast to other eukaryotes which usually express two closely related HMG1-like proteins, plant cells have multiple relatively variable proteins of this type. A systematic analysis of the DNA-binding properties of four chromosomal HMG domain proteins from maize revealed that they bind linear DNA with similar affinity. HMGa, HMGc1/2 and HMGd specifically recognise diverse DNA structures such as DNA mini-circles and supercoiled DNA. They induce DNA-bending, and constrain negative superhelical turns in DNA. In the presence of DNA, the HMG domain proteins can self-associate, whereas they are monomeric in solution. The maize HMG1-like proteins have the ability to facilitate the formation of nucleoprotein structures to different extents, since they can efficiently replace a bacterial chromatin-associated protein required for the site-specific beta-mediated recombination. A variable function of the HMG1-like proteins is indicated by their differential association with maize chromatin, as judged by their 'extractability' from chromatin with spermine and ethidium bromide. Collectively, these findings suggest that the various plant chromosomal HMG domain proteins could be adapted to act in different nucleoprotein structures in vivo.

Chromatin↗

PubMed2005

DNA knots reveal a chiral organization of DNA in phage capsids.

Icosahedral bacteriophages pack their double-stranded DNA genomes to near-crystalline density and achieve one of the highest levels of DNA condensation found in nature. Despite numerous studies, some essential properties of the packaging geometry of the DNA inside the phage capsid are still unknown. We present a different approach to the problems of randomness and chirality of the packed DNA. We recently showed that most DNA molecules extracted from bacteriophage P4 are highly knotted because of the cyclization of the linear DNA molecule confined in the phage capsid. Here, we show that these knots provide information about the global arrangement of the DNA inside the capsid. First, we analyze the distribution of the viral DNA knots by high-resolution gel electrophoresis. Next, we perform Monte Carlo computer simulations of random knotting for freely jointed polygons confined to spherical volumes. Comparison of the knot distributions obtained by both techniques produces a topological proof of nonrandom packaging of the viral DNA. Moreover, our simulations show that the scarcity of the achiral knot 4(1) and the predominance of the torus knot 5(1) over the twist knot 5(2) observed in the viral distribution of DNA knots cannot be obtained by confinement alone but must include writhe bias in the conformation sampling. These results indicate that the packaging geometry of the DNA inside the viral capsid is writhe-directed.

Bacteriophages↗

PubMed1983

Directional transport and integration of donor DNA in Haemophilus influenzae transformation.

DNA transport and integration in Haemophilus influenzae transformation was studied with a plasmid clone of homologous DNA (pCML6). Our results indicate that: (i) donor DNA enters specialized membranous extensions on the cell surface, which we have termed "transformasomes"; (ii) linear DNA undergoes degradation upon exiting transformasomes; and (iii) DNA without a free end remains within transformasomes and is not degraded. By comparing the fate of label from uniformly labeled versus middle-labeled DNA, it appears that donor DNA undergoes degradation from an end prior to recombining with the chromosome. Using donor DNA with covalently closed termini (hairpin ends) prevents efficient exit from transformasomes. When one hairpin is removed, exit of donor DNA is shown to be directional from the free end, with preferential homologous integration of the 3' strand from that end.

Base Sequence↗