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PubMed2005

Overview of mechanisms of cancer chemopreventive agents.

Epidemiological data provide evidence that it is possible to prevent cancer and other chronic diseases, some of which share common pathogenetic mechanisms, such as DNA damage, oxidative stress, and chronic inflammation. An obvious approach is avoidance of exposure to recognized risk factors. As complementary strategies, it is possible to render the organism more resistant to mutagens/carcinogens and/or to inhibit progression of the disease by administering chemopreventive agents. In a primary prevention setting, addressed to apparently healthy individuals, it is possible to inhibit mutation and cancer initiation by triggering protective mechanisms either in the extracellular environment or inside cells, e.g., by modifying transmembrane transport, modulating metabolism, blocking reactive species, inhibiting cell replication, maintaining DNA structure, modulating DNA metabolism and repair, and controlling gene expression. Tumor promotion can be counteracted by inhibiting genotoxic effects, favoring antioxidant and anti-inflammatory activity, inhibiting proteases and cell proliferation, inducing cell differentiation, modulating apoptosis and signal transduction pathways, and protecting intercellular communications. In a secondary prevention setting, when a premalignant lesion has been detected, it is possible to inhibit tumor progression via the same mechanisms, and in addition by affecting the hormonal status and the immune system in various ways, and by inhibiting tumor angiogenesis. Although tertiary prevention, addressed to cancer patients after therapy, is outside the classical definition of chemoprevention, it exploits similar mechanisms. It is also possible to affect cell-adhesion molecules, to activate antimetastasis genes, and to inhibit proteases involved in basement membrane degradation.

Anticarcinogenic Agents↗
PubMed2001

G-quadruplex DNA binding by a series of carbocyanine dyes.

We have examined a number of carbocyanine dyes for their ability to bind intramolecular G-quadruplex DNA structures (G4'-DNA) using a Taq polymerase stop assay. Of the five dyes examined, only one, N,N'-diethylthiacarbocyanine iodide (DTC), was found to bind to G4'-DNA. DTC was also the only dye found to inhibit human telomerase at 50 microM concentration.

Binding Sites↗
PubMed1989

Kinetic modeling of the recA protein promoted renaturation of complementary DNA strands.

Quantitative agarose gel assays reveal that the recA protein promoted renaturation of complementary DNA strands (phi X DNA) proceeds in two stages. The first stage results in the formation of unit-length duplex DNA as well as a distribution of other products ("initial products"). In the second stage, the initial products are converted to complex multipaired DNA structures ("network DNA"). In the presence of ATP, the initial products are formed within 2 min and are then rapidly converted to network DNA. In the absence of ATP, the initial products are formed nearly as fast as with ATP present, but they are converted to network DNA at a much lower rate. The time-dependent formation of initial products and network DNA from complementary single strands for both the ATP-stimulated and ATP-independent reactions can be modeled by using a simple two-step sequential kinetic scheme. This model indicates that the primary effect of ATP in the recA protein promoted renaturation reaction is not on the initial pairing step (which leads to the formation of initial products) but rather is to increase the rate at which subsequent pairing events can occur.

Adenosine Triphosphate↗
PubMed2005

Repeat instability: mechanisms of dynamic mutations.

Disease-causing repeat instability is an important and unique form of mutation that is linked to more than 40 neurological, neurodegenerative and neuromuscular disorders. DNA repeat expansion mutations are dynamic and ongoing within tissues and across generations. The patterns of inherited and tissue-specific instability are determined by both gene-specific cis-elements and trans-acting DNA metabolic proteins. Repeat instability probably involves the formation of unusual DNA structures during DNA replication, repair and recombination. Experimental advances towards explaining the mechanisms of repeat instability have broadened our understanding of this mutational process. They have revealed surprising ways in which metabolic pathways can drive or protect from repeat instability.

DNA Repair↗
PubMed2006

Dual role of MutS glutamate 38 in DNA mismatch discrimination and in the authorization of repair.

MutS plays a critical role in DNA mismatch repair in Escherichia coli by binding to mismatches and initiating repair in an ATP-dependent manner. Mutational analysis of a highly conserved glutamate, Glu38, has revealed its role in mismatch recognition by enabling MutS to discriminate between homoduplex and mismatched DNA. Crystal structures of MutS have shown that Glu38 forms a hydrogen bond to one of the mismatched bases. In this study, we have analyzed the crystal structures, DNA binding and the response to ATP binding of three Glu38 mutants. While confirming the role of the negative charge in initial discrimination, we show that in vivo mismatch repair can proceed even when discrimination is low. We demonstrate that the formation of a hydrogen bond by residue 38 to the mismatched base authorizes repair by inducing intramolecular signaling, which results in the inhibition of rapid hydrolysis of distally bound ATP. This allows formation of the stable MutS-ATP-DNA clamp, a key intermediate in triggering downstream repair events.

Adenosine Triphosphate↗
PubMed2001

Werner helicase relocates into nuclear foci in response to DNA damaging agents and co-localizes with RPA and Rad51.

BACKGROUND: Werner syndrome (WS) is an autosomal recessive disorder with many features of premature ageing. Cells derived from WS patients show genomic instability, aberrations in the S-phase and sensitivity to genotoxic agents. The gene responsible for WS (WRN) encodes a DNA helicase belonging to the RecQ helicase family. Although biochemical studies showed that the gene product of WRN (WRNp) interacts with proteins that participate in DNA metabolism, its precise biological function remains unclear. RESULTS: Using immunocytochemistry, we found that WRNp forms distinct nuclear foci in response to DNA damaging agents, including camptothecin (CPT), etoposide, 4-nitroquinolin-N-oxide and bleomycin. The presence of aphidicolin inhibited CPT-induced WRNp foci strongly but not bleomycin-induced foci. These WRNp foci overlapped with the foci of replication protein A (RPA) almost entirely and with the foci of Rad51 partially, implicating cooperative functions of these proteins in response to DNA damage. We also found that WRNp foci partially co-localize with sites of 5-bromo-2'-deoxy-uridine incorporation. CONCLUSIONS: These findings suggest that WRNp form nuclear foci in response to aberrant DNA structures, including DNA double-strand breaks and stalled replication forks. We propose that WRNp takes part in the homologous recombinational repair and in the processing of stalled replication forks.

4-Nitroquinoline-1-oxide↗
PubMed1996

Termination within oligo(dT) tracts in template DNA by DNA polymerase gamma occurs with formation of a DNA triplex structure and is relieved by mitochondrial single-stranded DNA-binding protein.

Xenopus laevis DNA polymerase gamma (pol gamma) exhibits low activity on a poly(dT)-oligo(dA) primer-template. We prepared a single-stranded phagemid template containing a dT41 sequence to test the ability of pol gamma to extend a primer through a defined oligo(dT) tract. pol gamma terminates in the center of this dT41 sequence. This replication arrest is abrogated by addition of single-stranded DNA-binding protein or by substitution of 7-deaza-dATP for dATP. These features are consistent with the formation of a T.A*T DNA triplex involving the primer stem. Replication arrest occurs under conditions that permit highly processive DNA synthesis by pol gamma. A similar replication arrest occurs for T7 DNA polymerase, which is also a highly processive DNA polymerase. These results suggest the possibility that DNA triplex formation can occur prior to dissociation of DNA polymerase. Primers with 3'-oligo(dA) termini annealed to a template with a longer oligo(dT) tract are not efficiently extended by pol gamma unless single-stranded DNA-binding protein is added. Thus, one of the functions of single-stranded DNA-binding protein in mtDNA maintenance may be to enable pol gamma to successfully replicate through dT-rich sequences.

Base Composition↗
PubMed1995

Imino proton exchange provides an 1H-NMR footprint of protein-DNA interactions: general strategy and application to the SRY HMG box.

A novel 1H nuclear magnetic resonance (NMR) strategy for "footprinting" specific protein-DNA target sites is demonstrated. Relative rates of site-specifc imino-proton exchange in the free and bound DNA duplex are determined by use of laminar-shifted shaped pulses in NOESY spectra. 2D exchange crosspeaks between imino (omega2 dimension) (omega1 dimension) resonances in principle provide site-specific probes of protein binding. Chemical exchange is distinguished from nuclear Overhauser enhancements(NOEs) to bound water by use of ROESY spectroscopy. This strategy is illustrated in 1H-NMR studies of the SRY high-mobility group (HMG) box, the Y-chromosome-encoded "master switch" for testis determination in man. In a specific complex between the protein and a 15-basepair DNA site, imino-proton exchange was observed to be damped selectively within the six basepair subsite 5'-ATTGTT, previously identified by random binding-site selection as an optimal SRY target sequence. The extent of damping correlates with sites of protein-DNA contacts in the minor groove but not with the magnitude of 1H-NMR complexation shifts. SRY binding has recently been shown to introduce significant distortions in DNA structure. The DNA is sharply bent and underwound; the minor groove is widened and major groove compressed. Our results demonstrate that despite such distortions base pairing is stably maintained. Protein binding in the DNA minor groove shields DNA imino protons from exchange with solvent.

Base Composition↗
PubMed2003

Efficiency of coaxial stacking depends on the DNA duplex structure.

Thermodynamic parameters of coaxial stacking at complementary helix-helix interfaces GX*pYG/CZVC (X,Y=A,C,T,G;*-nick) created by contiguous oligonucleotide hybridization were determined. The data obtained were compared to the thermodynamic parameters of coaxial stacking at the interfaces CX*pYC/GZVG. Multiple linear regression analysis has revealed that the free-energy increments of interaction for the contacts GX*pYG/CZVC and CX*pYC/GZVG can be described by a set of uniform Delta G degrees(X*pY/ZV) values. The difference in the observed free-energy of the coaxial stacking between the two sets is defined by the contribution from the factors reflecting structural differences between compared DNA duplexes.

Base Sequence↗
PubMed1990

Spectroscopic and calorimetric investigation on the DNA triplex formed by d(CTCTTCTTTCTTTTCTTTCTTCTC) and d(GAGAAGAAAGA) at acidic pH.

The equimolar mixture of d(CTCTTCTTTCTTTTCTTTCTTCTC) (dY24) and d(GAGAAGAAAGA) (dR11) [designated (dY24).(dR11)], forms at pH = 5 a DNA triplex, which mimicks the H-DNA structure. The DNA triplex was identified by the following criteria: (i) dY24 and dR11 co-migrate in a poly-acrylamide gel, with a mobility and a retardation coefficient comparable to those observed for an 11-triad DNA triplex, previously characterized in our laboratories (1); (ii) the intercalator ethidium bromide shows a poor affinity for (dR11).(dY24) at pH = 5, and a high affinity at pH = 8; (iii) the (dR11).(dY24) mixture is not a substrate for DNase I at pH = 5; (iv) the CD spectrum of (dR11).(dY24), at pH = 5, is consistent with those previously reported for triple-stranded DNA. The (dR11).(dY24) mixture exhibits a thermally induced co-operative transition, which appears to be monophasic, reversible and concentration dependent. This transition is attributed to the disruption of the DNA triplex into single strands. The enthalpy change of the triplex-coil transition was measured by DSC (delta Hcal = 129 +/- 6 kcal/mol) and, assuming a two-state model, by analysis of UV-denaturation curves (average of two methods delta HUV = 137 +/- 13 kcal/mol). Subtracting from delta Hcal of triplex formation the contributions due to the Watson-Crick helix and to the protonation of the C-residues, we found that each pyrimidine binding into the major groove of the duplex, through a Hoogsteen base pair, is accompanied by an average delta H = -5.8 +/- 0.6 kcal/mol. The effect on the stability of the (dR11).(dY24) triplex due to the substitution of a T:A:T triad with a T:T:T one was also investigated.

Base Sequence↗
PubMed1997

Molecular and cytogenetic analyses of stably and unstably expressed transgene loci in tobacco.

To study the influence of genomic context on transgene expression, we have determined the T-DNA structure, flanking DNA sequences, and chromosomal location of four independent transgene loci in tobacco. Two of these loci were stably expressed in the homozygous condition over many generations, whereas the other two loci became unstable after several generations of homozygosity. The stably expressed loci comprised relatively simple T-DNA arrangements that were flanked on at least one side by plant DNA containing AT-rich regions that bind to nuclear matrices in vitro. Of the unstably expressed loci, one consisted of multiple incomplete T-DNA copies, and the second contained a single intact T-DNA; in both cases, however, binary vector sequences were directly contiguous to a right T-DNA border. Fluorescence in situ hybridization demonstrated that the two stably expressed inserts were present in the vicinity of telomeres. The two unstably expressed inserts occupied intercalary and paracentromeric locations, respectively. Results on the stability of transgene expression in F1 progeny obtained by intercrossing the four lines and the sensitivity of the four transgene loci to inactivation in the presence of an unlinked "trans-silencing" locus are also presented. The findings are discussed in the context of repetitive DNA sequences and the allotetraploid nature of the tobacco genome.

Amino Acid Sequence↗
PubMed2001

Topoisomerase III acts upstream of Rad53p in the S-phase DNA damage checkpoint.

Deletion of the Saccharomyces cerevisiae TOP3 gene, encoding Top3p, leads to a slow-growth phenotype characterized by an accumulation of cells with a late S/G2 content of DNA (S. Gangloff, J. P. McDonald, C. Bendixen, L. Arthur, and R. Rothstein, Mol. Cell. Biol. 14:8391-8398, 1994). We have investigated the function of TOP3 during cell cycle progression and the molecular basis for the cell cycle delay seen in top3Delta strains. We show that top3Delta mutants exhibit a RAD24-dependent delay in the G2 phase, suggesting a possible role for Top3p in the resolution of abnormal DNA structures or DNA damage arising during S phase. Consistent with this notion, top3Delta strains are sensitive to killing by a variety of DNA-damaging agents, including UV light and the alkylating agent methyl methanesulfonate, and are partially defective in the intra-S-phase checkpoint that slows the rate of S-phase progression following exposure to DNA-damaging agents. This S-phase checkpoint defect is associated with a defect in phosphorylation of Rad53p, indicating that, in the absence of Top3p, the efficiency of sensing the existence of DNA damage or signaling to the Rad53 kinase is impaired. Consistent with a role for Top3p specifically during S phase, top3Delta mutants are sensitive to the replication inhibitor hydroxyurea, expression of the TOP3 mRNA is activated in late G1 phase, and DNA damage checkpoints operating outside of S phase are unaffected by deletion of TOP3. All of these phenotypic consequences of loss of Top3p function are at least partially suppressed by deletion of SGS1, the yeast homologue of the human Bloom's and Werner's syndrome genes. These data implicate Top3p and, by inference, Sgs1p in an S-phase-specific role in the cellular response to DNA damage. A model proposing a role for these proteins in S phase is presented.

Alkylating Agents↗
PubMed2004

Meiotic recombination intermediates and mismatch repair proteins.

Mismatch repair proteins are a highly diverse group of proteins that interact with numerous DNA structures during DNA repair and replication. Here we review data for the role of Msh4, Msh5, Mlh1, Mlh3 and Exo1 in crossing over. Based on the paradigm of interactions developed from studies of mismatch repair, we propose models for the mechanism of crossover implementation by Msh4/Msh5 and Mlh1/Mlh3.

Animals↗
PubMed1994

Metabolic activation routes of arylamines and their genotoxic effects.

Two different types of DNA adducts are formed from many aromatic amines by bioactivation: N-acetylated and nonacetylated, arylamine DNA adducts. It has become clear from experiments using N-acetyl-2-aminofluorene and 2-aminofluorene adducts to C8 of deoxyguanosine that these two types of adducts may have different effects on DNA structure and DNA replication. We have determined blocking of DNA replication by various other N-acetylarylamine and arylamine deoxyguanosine adducts. It was found that the N-acetyl group in general is required for blocking of DNA replication; the nature of the aromatic moiety seems to be of minor importance. Little information is available on the genotoxic effects of these adducts in mammalian cells in vivo. We have tried to get more insight in this by investigating the clastogenicity, the initiation of preneoplastic cells, and the promotional effects of various aromatic amines from which different ratios of N-acetylarylamine DNA adducts to arylamine DNA adducts are formed in the rat liver. Our results show that formation of N-acetylarylamine adducts to C8 of deoxyguanosine in the liver is correlated with clastogenicity and hepatic promoting effect. Initiation capacities, however, seem to be correlated with formation of nonacetylated, arylamine adducts. Mechanisms by which formation of N-acetylarylamine DNA adducts may generate a promoting effect in the liver are discussed.

Amines↗
PubMedDate not supplied

[Methylation of adenine residues in DNA of eukaryotes].

Like in bacteria, DNA in these organisms is subjected to enzymatic modification (methylation) both at adenine and cytosine residues. There is an indirect evidence that adenine DNA methylation takes place also in animals. In plants m6A was detected in total, mitochondrial and nuclear DNAs; in plants one and the same gene (DRM2) can be methylated both at adenine and cytosine residues. ORF homologous to bacterial adenine DNA-methyltransferases are present in nuclear DNA of protozoa, yeasts, insects, nematodes, higher plants, vertebrates and other eukaryotes. Thus, adenine DNA-methyltransferases can be found in the various evolutionary distant eukaryotes. First N6-adenine DNA-methyltransferase (wadmtase) of higher eukaryotes was isolated from vacuolar fraction of vesicles obtained from aging wheat coleoptiles; in the presence of S-adenosyl-L-methionine this Mg2+ -, Ca2+ -dependent enzyme de novo methylates first adenine residue in TGATCA sequence in single- and double-stranded DNA but it prefers single-stranded DNA structures. Adenine DNA methylation in eukaryotes seems to be involved in regulation of both gene expression and DNA replication including replication of mitochondrial DNA. It can control persistence of foreign DNA in a cell and seems to be an element of R-M system in plants. Thus, in eukaryotic cell there are, at least, two different systems of the enzymatic DNA methylations (adenine and cytosine ones) and a special type of regulation of gene functioning based on the combinatory hierarchy of these interdependent genome modifications.

Adenine↗
PubMed2006

[Study of DNA damage induced by potassium dichromate and glutathione with atomic force microscope].

OBJECTIVE: To observe calf thymus DNA damage induced by potassium dichromate in combination with glutathione (GSH). METHODS: Atomic force microscope and ultraviolet spectrum (UV) were used to observe the alterations of the DNA ultrastructure and absorption spectrum. RESULTS: Atomic force microscopy revealed no breaks of the DNA strand in response to treatment with potassium dichromate alone, but when coupled with GSH at proper concentrations, potassium dichromate induced alterations in the DNA structure and DNA fragmentation. UV examination also confirmed these findings by showing increased absorption intensity of the maximum UV peak following combined treatment of the DNA with potassium dichromate and GSH. CONCLUSION: These morphological and spectrographic evidences verified the important role of GSH in mediating the generation of various tumor-inducing intermediate products of potassium dichromate.

Animals↗