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PubMed1992

Chemical and biochemical postlabeling methods for singling out specific oxidative DNA lesions.

A survey of the main available chemical and biochemical postlabeling assays for measuring oxidative DNA damage is reported. Two main approaches, radio and fluorescent postlabeling, have been used in order to reach a high level of sensitivity of detection. This is required for the measurement of DNA damage within cells and tissues upon exposure to agents of oxidative stress. Most of the methods are based on liquid chromatographic separation of defined DNA modifications following either acidic hydrolysis or enzymic digestion of DNA. In a subsequent step, the isolated base or sugar damages are either radiolabeled or made fluorescent by chemical or enzymatic reactions. Emphasis is placed on the recently developed high performance liquid chromatographic 32P-postlabeling assay, which allows the specific and sensitive measurement of various base damages including adenine N-1 oxide and 5-hydroxymethyluracil at the level of one modification per 10(7) normal bases in a sample size of 1 microgram of DNA. Examples of application of radioactive postlabeling to the measurement of DNA base damage following exposure of human cells to oxidizing agents including hydrogen peroxide and UVA radiation are provided.

Adenosine Triphosphate↗
PubMedDate not supplied

Role of the central metal ion and ligand charge in the DNA binding and modification by metallosalen complexes.

Several metal complexes of three different functionalized salen derivatives have been synthesized. The salens differ in terms of the electrostatic character and the location of the charges. The interactions of such complexes with DNA were first investigated in detail by UV-vis absorption titrimetry. It appears that the DNA binding by most of these compounds is primarily due to a combination of electrostatic and other modes of interactions. The melting temperatures of DNA in the presence of various metal complexes were higher than that of the pure DNA. The presence of additional charge on the central metal ion core in the complex, however, alters the nature of binding. Bis-cationic salen complexes containing central Ni(II) or Mn(III) were found to induce DNA strand scission, especially in the presence of co-oxidant as revealed by plasmid DNA cleavage assay and also on the basis of the autoradiogram obtained from their respective high-resolution sequencing gels. Modest base selectivity was observed in the DNA cleavage reactions. Comparisons of the linearized and supercoiled forms of DNA in the metal complex-mediated cleavage reactions reveal that the supercoiled forms are more susceptible to DNA scission. Under suitable conditions, the DNA cleavage reactions can be induced either by preformed metal complexes or by in situ complexation of the ligand in the presence of the appropriate metal ion. Also revealed was the fact that the analogous complexes containing Cu(II) or Cr(III) did not effect any DNA strand scission under comparable conditions. Salens with pendant negative charges on either side of the precursor salicylaldehyde or ethylenediamine fragments did not bind with DNA. Similarly, metallosalen complexes with net anionic character also failed to induce any DNA modification activities.

Autoradiography↗
PubMed1993

Use of repair endonucleases to characterize DNA damage induced by reactive oxygen species in cellular and cell-free systems.

A number of repair endonuclease, viz. endonuclease III, formamidopyrimidine-DNA glycosylase (FPG protein), endonuclease IV, exonuclease III and UV endonuclease, is used to simultaneously quantify various types of DNA modifications, which were induced by agents that generate reactive oxygen species. Under cell-free conditions, two types of DNA damage profiles are obtained. The profiles induced by chemically generated singlet oxygen and by various photosensitizers (acridine orange, methylene blue, riboflavin, hematoporphyrin) plus light are dominated by base modifications sensitive to FPG protein, while 5,6-dihydropyrimidines (recognized by endonuclease III), sites of base loss (AP sites, recognized by endonuclease IV and exonuclease III) and strand breaks are minor lesions. In contrast, the DNA damage profile induced by hydroxyl radicals (gamma-rays) consists of approx. equal levels of base modifications. AP sites and strand breaks. The damage profiles induced by Fe(III)-EDTA in the presence of superoxide and by Fe(III)-nitrilotriacetate in the presence of H2O2 do not differ from that by hydroxyl radicals. The damage profile induced by Cu(II)-phenanthroline deviates by high levels of AP sites that are recognized by endonuclease IV and exonuclease III-but not by those AP endonucleases which cleave at the 3' site-and probably represent AP sites oxidized at C-1'. The damage induced by Fe(III)-bleomycin plus H2O2 deviates by an increased level of double strand breaks and the absence of endonuclease-sensitive base modifications. Cellular DNA damage profiles are obtained from bacteria, cultured mammalian cells and mammalian mitochondria after exposure to acridine orange plus visible light. A comparison with the cell-free profiles reveals that the damage in all three systems is not induced indirectly by hydroxyl radicals or an activation of cellular nucleases, but by the same mechanism that is responsible for the cell-free DNA damage.

Acridine Orange↗
PubMed1993

Application of high-performance liquid chromatography for recognition of covalent nucleic acid modification with anticancer drugs.

Covalent modification of DNA by antineoplastic agents represents a potent biochemical lesion which can play a major role in drug mechanism of action. The ability to measure levels of DNA covalent modifications in target cells in vivo may, therefore, be seen as the ultimate form of therapeutic drug monitoring. Additionally, elucidation of the structure of critical DNA adducts and definition of their role in tumour cell cytotoxicity will provide more selective targets for rational drug design of new cancer chemotherapeutic agents. High-performance liquid chromatography has contributed significantly to all these areas. In vivo levels of nucleic acid covalent modifications are in the range of 1 in 10(5)-10(8) nucleotides precluding the use of conventional high-performance liquid chromatographic detection methods. Several classes of natural product anticancer drugs have been shown to bond covalently to nucleic acids under optimal laboratory conditions. These have proved more accessible to high-performance liquid chromatographic analysis because of their lipophilicity and strong UV chromophores. However, the majority of experimental evidence to date suggests that with the exception of mitomycin C and morpholino-anthracyclines these compounds do not exert their primary mechanism of action through nucleic acid covalent modification. DNA adducts of alkylating and platinating agents are more difficult to detect by high-performance liquid chromatography and can be chemically unstable. These compounds interact with DNA on the basis of chemical kinetics. Thus, the principle sites of attachment tend to be with the most nucleophilic base (guanine) at its most reactive centre (N-7 position). Limited in vivo high-performance liquid chromatographic studies with all classes of anticancer drugs indicate a much more complex pattern of adductation than would have been anticipated from in vitro studies alone. Some of these differences are probably due to methodological artefacts but these studies stress the need for sensitive detection methods and reliable sample preparation (nucleic acid extraction and digestion techniques) when attempting to determine nucleic acid covalent modifications by anticancer drugs.

Animals↗
PubMed1989

Site-directed chemical modification for probing DNA-protein interactions. Osmium tetroxide modification of the -10 site of the lacUV5 promoter enhances open complex formation.

A new experimental approach, site-directed chemical modification, was used to explore relationships between RNA polymerase-promoter interactions and function. For this study, the lacUV5 promoter with an exposed -10 thymine on the non-template strand was constructed. Osmium tetroxide was selected as the thymine modifying reagent. Modification occurred predominantly at the exposed -10 T with 5-fold less reactivity at the -12 T residue. The isolated modified strand was used to reconstitute a lacUV5 promoter with -10 (-12) adducts. OsO4 modification at both the -10 and -12 positions of the lacUV5 promoter significantly enhances Escherichia coli RNA polymerase-promoter open complex formation relative to the unmodified promoter. DNase I cleavage sites at -7, -8, and -10 of the unmodified promoter were rendered insusceptible to scission in the modified promoter. However, no difference can be detected in the RNA polymerase footprints for unmodified versus modified open complexes. The latter are fully capable of productive transcription with comparable amounts of identical run-off transcripts to unmodified open complexes. A 16 degrees C reduction in Tm was found for a 14-base pair oligonucleotide duplex containing a single OsO4-bispyridine adduct. The latter result suggests that open complex formation appears to be enhanced due to promoter unpairing at the -10 (-12) adduct sites.

Base Sequence↗
PubMed1996

Modulation of non-templated nucleotide addition by Taq DNA polymerase: primer modifications that facilitate genotyping.

Taq DNA polymerase can catalyze non-templated addition of a nucleotide (principally adenosine) to the 3' end of PCR-amplified products. Recently, we showed that this activity, which is primer-specific, presents a potential source of error in genotyping studies based on the use of short tandem repeat (STR) markers. Furthermore, in reviewing our data, we found that non-templated nucleotide addition adjacent to a 3' terminal C is favored and that addition adjacent to a 3' terminal A is not. It was clear, however, that features of the template in addition to the 3' terminal base also affect the fraction of product adenylated. To define consensus sequences that promote or inhibit product adenylation, we transplanted sequences between the 5' ends of the reverse primers of markers that are adenylated and those of markers that are not adenylated. It proved difficult to identify a single sequence capable of protecting the products of all markers from non-templated addition of nucleotide. On the other hand, placing the sequence GTTTCTT on the 5' end of reverse primers resulted in nearly 100% adenylation of the 3' end of the forward strand. This modification or related ones (called "PIG-tailing") should facilitate accurate genotyping and efficient T/A cloning.

Base Sequence↗
PubMed1979

Characterization of DNA damages by filtration through nitrocellulose filters: a simple probe for DNA-modifying agents.

A simple technique for the detection of DNA-modifying agents is described. The double-stranded covalently closed circular DNA of phage PM2 is exposed to the modifying agent and then analysed for DNA damages by assays involving only incubation steps and filtration through nitrocellulose filters. The technique described allows the measurement of DNA modifications which lead to local denaturation of the DNA double helix, interstrand cross-links, single- and double-strand breaks, damages which render the phosphodiester bonds of the DNA sensitive to hydrolysis and damages which labilise the glycosylic bond between base and sugar moiety.

DNA, Circular↗
PubMedDate not supplied

S1 nuclease mapping of the phage Mu mom gene promoter: a model for the regulation of mom expression.

The mom gene of bacteriophage Mu encodes a DNA modification function. Expression of this modification requires the host Escherichia coli Dam (DNA-adenine methylase) function and the transacting phage Mu Dad function. The mom gene was subcloned into a variety of sites on plasmid pBR322. Insertions were made into the HincII and PvuI sites within the amp gene and into the ClaI site of the tet gene promoter. The only clones found were those in which the orientation of the mom gene prevents its transcription from the vector promoter(s), suggesting that constitutive expression of mom from a foreign promoter can occur independently of Dad function but is lethal for the cell. Employing S1 nuclease mapping, we have identified two Mu mRNA transcripts: (1) the gin transcript extends into the gin-mon intercistronic divide and terminates downstream from the BclI site; and (2) the mom transcript appears to initiate about 74 bp upstream from the BclI site, 12 bp downstream from a promoter-like sequence. Production of the mom transcript is dependent on the host Dam activity and on Dad transactivation. In contrast, the gin transcript is produced independently of Dam and Dad functions; the gin transcript may extend into the mom gene, but it appears to be either degraded at the 3' end or differentially terminated. We propose that regulation of mom gene transcription involves both positive and negative regulatory proteins, and that binding of the Dad protein (a "late" Mu protein) is required for transcription initiation by the host RNA polymerase. However, Dad protein action may be inhibited by prior binding of a repressor to the mom operator, located farther upstream. We propose that this repressor (encoded by a phage or host gene) binds to the operator only when there is no active Dam enzyme present, i.e., when there is no methylation of (or methylase binding to) the G-A-T-C sites within the mom operator.

Base Sequence↗
PubMed2004

The PCNA-RFC families of DNA clamps and clamp loaders.

The proliferating cell nuclear antigen PCNA functions at multiple levels in directing DNA metabolic pathways. Unbound to DNA, PCNA promotes localization of replication factors with a consensus PCNA-binding domain to replication factories. When bound to DNA, PCNA organizes various proteins involved in DNA replication, DNA repair, DNA modification, and chromatin modeling. Its modification by ubiquitin directs the cellular response to DNA damage. The ring-like PCNA homotrimer encircles double-stranded DNA and slides spontaneously across it. Loading of PCNA onto DNA at template-primer junctions is performed in an ATP-dependent process by replication factor C (RFC), a heteropentameric AAA+ protein complex consisting of the Rfc1, Rfc2, Rfc3, Rfc4, and Rfc5 subunits. Loading of yeast PCNA (POL30) is mechanistically distinct from analogous processes in E. coli (beta subunit by the gamma complex) and bacteriophage T4 (gp45 by gp44/62). Multiple stepwise ATP-binding events to RFC are required to load PCNA onto primed DNA. This stepwise mechanism should permit editing of this process at individual steps and allow for divergence of the default process into more specialized modes. Indeed, alternative RFC complexes consisting of the small RFC subunits together with an alternative Rfc1-like subunit have been identified. A complex required for the DNA damage checkpoint contains the Rad24 subunit, a complex required for sister chromatid cohesion contains the Ctf18 subunit, and a complex that aids in genome stability contains the Elg1 subunit. Only the RFC-Rad24 complex has a known associated clamp, a heterotrimeric complex consisting of Rad17, Mec3, and Ddc1. The other putative clamp loaders could either act on clamps yet to be identified or act on the two known clamps.

DNA↗
PubMed1997

Enzymatic recognition and biological effects of photodynamic damage induced in DNA by 1,6-dioxapyrene plus UVA.

The specific recognition of DNA modifications by repair endonucleases was used to characterize DNA damage induced by 1,6-dioxapyrene (1,6-DP) in the presence of ultraviolet light at 365 nm (UVA) in the plasmid YEplac181. Under cell free conditions, 1,6-DP plus UVA generated lesions are recognized by the UvrABC endonuclease, the proteins Nth, Nfo and Fpg. The number of UvrABC sensitive sites was at least ten-fold higher than that of Fpg or Nth sensitive sites. Moreover, 1,6-DP plus UVA generated single-strand breaks which are the second most frequent lesions. To investigate the biological effect of DNA damage, YEplac181 DNA was treated with 1,6-DP plus UVA and transformed into Escherichia coli or Saccharomyces cerevisiae. In Escherichia coli, the transformation efficiency of 1,6-DP plus UVA treated DNA was greatly reduced in the uvrA mutant compared to that in the wild-type strain. However, the transforming efficiency was not affected in Fpg-deficient strains. In Saccharomyces cerevisiae, the transformation efficiency of 1,6-DP plus UVA treated YEplac181 was greatly reduced in the rad14::URA3 strain. The photobiological effect of 1,6-DP plus UVA was also analysed in haploid yeast strains of various repair capacities. The results show that the yeast strain defective in the nucleotide excision repair pathway (rad14::URA3) is hypersensitive to 1,6-DP plus UVA treatment as compared to the parental wild-type strain. It is confirmed that the lethal effect of 1,6-DP plus UVA on wild-type yeast is strongly oxygen dependent, whereas the survival of the rad14::URA3 mutant only exhibits a minor oxygen dependence. To conclude, our data show that the photodynamic DNA lesions induced by 1,6-DP plus UVA can be recognized and repaired in pro- and eukaryotic cells by the nucleotide excision repair pathway.

Carbon-Oxygen Lyases↗
PubMed1991

DNA adducts, detected by 32P-postlabelling, in the foregut of patients with familial adenomatous polyposis and in unaffected controls.

Duodenal neoplasms in patients with familial adenomatous polyposis (FAP) cluster around the ampulla; duodenal neoplasia is common but gastric neoplasia is rare. These observations suggest that bile enhances neoplasia by carrying carcinogens to target cells. A critical step in carcinogenesis is the reaction of carcinogens with DNA to form chemical adducts. Using 32P-postlabelling to measure DNA adducts we asked: (i) are there more adducts in the duodenum of patients with FAP than in the duodenum of normal patients? (ii) Are there more adducts in the duodenum than in the stomach? We measured adducts in duodenal biopsies from 51 patients with FAP and 30 age-matched controls; and paired gastric and duodenal biopsies from 31 FAP patients and six controls. The foregut of 90% of all patients studied contained DNA modifications with characteristics of aromatic non-polar DNA adducts. In duodenal biopsies, adduct labelling per 10(9) DNA nucleotides was significantly higher in FAP patients (median 15.0, range 0-162) than in normal patients (median, 7.5, range 0-40; P = 0.0002). In paired duodenal and gastric biopsies from 31 FAP patients, adduct labelling was significantly higher in duodenal DNA (median 15, range 0-109) than in stomach DNA (median 8.0, range 0-40; P = 0.0004). Age, gender or smoking did not account for these differences. Gastric adducts (median 3.5, range 0-10) were lower than duodenal adducts (median 8.5, range 0-29) in paired biopsies from six control patients, P = 0.031). These results support the hypothesis that pancreaticobiliary secretions may be involved in the pathogenesis of foregut neoplasia in FAP.

Adenomatous Polyposis Coli↗
PubMed1992

Effects of DNA looping on pyrimidine dimer formation.

We have assessed the effects of DNA curvature on pyrimidine dimer (PD) formation by examining the pattern of PD formation in DNA held in a loop by lambda repressor. The loop region was composed of diverse DNA sequences such that potential PD sites occurred throughout the loop. PD formation in the loop occurred with peaks at approximately 10 base intervals, just 3' of where the bending of the DNA was inferred to be toward the major groove. This relationship between the peaks and the DNA curvature is essentially identical to that observed in the nucleosome. This indicates that DNA curvature is the major source of the periodicity of PD formation in the nucleosome, and supports an earlier model of the conformation of nucleosomal DNA based on PD formation. DNA loops containing diverse sequences should be of general value for assessing the effects of DNA curvature on DNA modification by other agents used to probe DNA-protein interactions and DNA conformation.

Base Sequence↗
PubMed1989

Basis for changes in DNA recognition by the EcoR124 and EcoR124/3 type I DNA restriction and modification enzymes.

EcoR124 and EcoR124/3 are type I DNA restriction and modification systems. The EcoR124/3 system arose from the EcoR124 system some 15 years ago and at the electron microscopic DNA heteroduplex level the genes for both systems are still apparently identical. We have shown that the DNA sequences recognized by the two systems are GAA(N6)RTCG for EcoR124 and GAA(N7)RTCG for EcoR124/3. The sequences thus differ only in the length of the non-specific spacer. This difference nevertheless places the two specific domains of the EcoR124/3 recognition sequence 0.34 nm further apart and rotates them 36 degrees with respect to those of EcoR124, which implies major structural differences in the proteins recognizing these sequences. We have now determined the nucleotide sequences of the hsdS and hsdM genes of both systems and of the hsdR gene of EcoR124/3. The hsdS gene products provide DNA sequence specificity in both restriction and modification, the hsdM gene products are necessary for modification and all three hsd gene products are required for restriction. The only difference that we have detected between the two systems is that a 12 base-pair sequence towards the middle of the hsdS gene is repeated twice in the EcoR124 gene and three times in the EcoR124/3 gene. We have deleted one of the repeats in the EcoR124/3 gene and shown that this changes the specificity to that of EcoR124. Thus, the extra four amino acids in the middle of the EcoR124/3 hsdS gene product, which in an alpha-helical configuration would extend 0.6 nm, are sufficient to explain the differences in sequence recognition. We suggest that the EcoR124/3 system was generated by an unequal crossing over and argue that this kind of specificity change should not be rare in Nature.

Amino Acid Sequence↗
PubMed1996

DNA modifying agents as tools for studying chromatin structure.

DNA modification leads to mutagenicity, carcinogenicity, and cytotoxicity. Because the majority of cellular DNA exists as chromatin, histone and other proteins may mediate DNA binding and bonding reactions in vivo. Chromatin modification by a wide variety of agents, including intercalators, groove binders, cross-linkers, and strand cleavers, is reviewed. Although the majority of agents demonstrate a preference for linker over core DNA, the sequence preference appears to be retained in most cases, suggesting that both the major and minor grooves in chromatin are quite accessible to small molecules. Furthermore, only a handful of agents demonstrate the 10-11 base pair periodicity of hydroxyl radical, strongly supporting a dynamic model of nucleosome structure.

Animals↗
PubMed1999

What triggers senescence in Podospora anserina?

Senescence of Podospora anserina is triggered by a cytoplasmic and infectious factor (the determinant of senescence) and is always correlated with mitochondrial DNA modifications, especially with the accumulation of small circular subgenomic DNA molecules, the senDNAs. Several observations have suggested that the senDNAs could be the cytoplasmic and infectious determinant. However, we show here (1) that senDNA molecules can be transferred to a young culture without the cotransmission of the determinant of senescence and (2) that the determinant of senescence does not segregate as a mitochondrial DNA mutation. Overall, our data strongly argue that amplification of senDNA molecules in the mitochondria is not an intrinsic property of these small DNA molecules. They question the nature of the actual determinant of senescence.

Aging↗
PubMed1992

Photochemistry of nucleic acids in cells.

A survey of the recent aspects of the main photoreactions induced by far-UV radiation in cellular DNA is reported. This mostly includes the formation of cyclobutadipyrimidines, pyrimidine(6-4)pyrimidone photoadducts and related Dewar valence isomers in various eukaryotic and prokaryotic cells, as monitored by using either specific or more general assays. Information is also provided on mechanistic aspects regarding the formation of 5,6-dihydro-5-(alpha-thyminyl) thymine, the so-called "spore photoproduct" within far-UV-irradiated bacterial spores. The second major topic of the review deals with the effects of near-UV radiation and visible light on cellular DNA which are mostly mediated by photosensitizers. The main photoreactions of furocoumarins with DNA, one major class of photosensitizers used in the phototherapy of skin diseases, involve a [2 + 2] cycloaddition to the thymine bases according to an oxygen-independent mechanism. In contrast a second type of photosensitized reaction which appears to play a major role in the genotoxic effects of both near-UV and visible light requires the presence of oxygen. The photodynamic effects which are mediated by either still unidentified endogenous photosensitizers or defined exogenous photosensitizers lead to the formation of a wide spectrum of DNA modifications including base damage, oligonucleotide strand breaks and DNA-protein cross-links.

Animals↗
PubMed2004

Advances in chromatin remodeling and human disease.

Epigenetic factors alter phenotype without changing genotype. A primary molecular mechanism underlying epigenetics is the alteration of chromatin structure by covalent DNA modifications, covalent histone modifications, and nucleosome reorganization. Remodeling of chromatin structure regulates DNA methylation, replication, recombination, and repair as well as gene expression. As these functions would predict, dysfunction of the proteins that remodel chromatin causes an array of multi-system disorders and neoplasias. Insights from these diseases suggest that during embryonic and fetal life, environmental distortions of chromatin remodeling encode a 'molecular memory' that predispose the individual to diseases in adulthood.

Acetylation↗