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PubMed1985

Interaction of EcoRII restriction and modification enzymes with synthetic DNA fragments. V. Study of single-strand cleavages.

Concatemer DNA duplexes which contain at the EcoRII restriction endonuclease cleavage sites (formula; see text) phosphodiester, phosphoamide or pyrophosphate internucleotide bonds have been synthesized. It has been shown that this enzyme did not cleave the substrate at phosphoamide bond. EcoRII endonuclease catalyzes single-strand cleavages both in dA- and dT-containing strands of the recognition site if the cleavage of the other strand has been blocked by modification of scissile bond or if the other strand has been cleaved. This enzyme interacts with both strands of the DNA recognition site, each of them being cleaved independently on the cleavage of another one. Nucleotide sequences flanking the EcoRII site on both sides are necessary for effective cleavage of the substrate.

Base Sequence↗

PubMed1987

The effect of platinum derivatives on interfacial properties of DNA.

The interaction of DNA modified by the binding of various platinum compounds with an electrically charged mercury surface was studied by means of linear sweep voltammetry. It was found that DNA and its adducts with antitumour active cis-diammine-dichloroplatinum(II) (cis-DDP) on the one hand and antitumour inactive trans-diamminedichloroplatinum(II) (trans-DDP) and diethylenetriaminechloroplatinum(II) chloride (dien-Pt) on the other were unwound due to their adsorption on the negatively charged mercury surface polarized to the potentials of a narrow region around -1.2 V (vs. saturated calomel electrode). The modification of DNA by bifunctional platinum compounds (cis- and trans-DDP) resulted in a substantial lowering of the extent of this interfacial conformational rearrangement, the modification by trans-DDP being more effective. The modification of DNA by monofunctional dien-Pt influenced the unwinding of DNA on the mercury surface only negligibly. It has been concluded that in particular interstrand cross-links induced by platinum compounds in DNA are responsible for the effect of these drugs on the extent of the interfacial unwinding of DNA. This conclusion is in good agreement with the view that among the lesions induced in DNA by platinum compounds, the interstrand cross-links are of less significance from the point of view of the antitumour efficacy of these inorganic drugs.

Antineoplastic Agents↗

PubMed2005

Coordinated changes in DNA methylation and histone modifications regulate silencing/derepression of luteinizing hormone receptor gene transcription.

We have previously demonstrated that transcription of the luteinizing hormone receptor (LHR) gene is subject to repression by histone deacetylation at its promoter region, where a histone deacetylase (HDAC)/mSin3A complex is anchored at a proximal Sp1 site. The present studies have shown that epigenetic silencing and activation of the LHR gene is achieved through coordinated regulation at both the histone and DNA levels. The HDAC inhibitor trichostatin A (TSA) evoked robust but significantly lower activation of the LHR gene in JAR than in MCF-7 cells. This effect was localized to the 176-bp promoter region, which is highly methylated in JAR and lightly methylated in MCF-7 cells. Consequently, TSA and the DNA demethylating reagent 5-azacytidine (5-AzaC) caused marked synergistic activation of the LHR gene in JAR but not in MCF-7 cells. Multiple site-specific lysine acetylation of H3/H4 is associated with such LHR gene activation. Methylation or acetylation of H3 at K9 is present at the silenced and derepressed LHR promoter, respectively. While DNA methylation levels did not affect the histone code of the LHR gene promoter, demethylation of the promoter CpG sites was necessary for maximal stimulation of this gene. Mechanistically, the combined actions of TSA and 5-AzaC, but not either 5-AzaC or TSA alone, resulted in complete demethylation of the LHR gene promoter in JAR cells. Release of the repressive HDAC/mSin3A complex from the LHR gene promoter in both cell types required both TSA-induced changes of histone modifications and, concurrently, a demethylated promoter. Also, Dnmt1 was largely dissociated from the LHR gene promoter in the presence of TSA or TSA plus 5-AzaC, and binding of MBD2 in JAR cells was diminished upon conversion of the promoter to a demethylated state. Such changes induced a more permissive chromatin where recruitment of polymerase II and TFIIB to the promoter was significantly increased. The activated state of the LHR gene induced by TSA and 5-AzaC in JAR and MCF-7 cells was observed basally in LHR-expressing PLC cells, in which the promoter is unmethylated and associated with hyperacetylated histones. Consequently, PLC cells are unresponsive to drug treatment. These findings have elucidated a regulatory mechanism whereby concurrent dissociation of repressors and association of activators and basal transcriptional components, resulting from coordinated histone hyperacetylation and DNA demethylation, lead to derepression of the LHR gene expression.

Acetylation↗

PubMed1997

Covalent attachment of ethidium to DNA results in enhanced topoisomerase II-mediated DNA cleavage.

The classic DNA intercalator, ethidium, was used to probe the effects of (i) intercalation and (ii) covalent modification of the DNA on the catalytic activity of topoisomerase II. Ethidium bromide, which binds reversibly to DNA via intercalation, does not stimulate topoisomerase II-mediated DNA cleavage at concentrations up to 100 microM, indicating that the intercalative binding of this molecule to DNA is not sufficient to alter the activity of the enzyme. In contrast, covalent attachment of the photoreactive ethidium analog to DNA resulted in marked enhancement of topoisomerase II-mediated single- and double-stranded DNA cleavage. This increase in DNA cleavage was observed at very low drug binding densities (<1 drug per 10-80 base pairs) which correspond to nanomolar concentrations, as compared with other topoisomerase II poisons such as etoposide or m-AMSA which require micromolar concentrations to elicit comparable DNA cleavage levels. Over the past decade, topoisomerase II has been an important target for a variety of clinically relevant anticancer agents due to the abilities of these agents to convert this enzyme to a cellular toxin resulting in an increase in the levels of enzyme-mediated DNA breaks. Modification of DNA by covalently attaching a DNA-targeting intercalating agent (i.e., ethidium bromide) resulted in a marked shift of the cleavage/religation equilibrium of the enzyme toward the cleaved state "poison" topoisomerase II as observed by the enhancement in single- and double-stranded cleavage; thus, key insight was gained into the mechanism(s) through which DNA binding agents may influence the catalytic properties of topoisomerase II. These data demonstrate that conversion of a reversible ethidium-DNA complex to an irreversible adduct results in the transformation of an ineffective intercalating drug into a potent topoisomerase II-targeted agent. Finally, they provide support for the recently proposed "positional poisoning model" for the actions of DNA lesions and anticancer drugs on the type II enzyme.

Animals↗

PubMed1977

Carbodiimide modification of superhelical PM2 DNA: considerations regarding reaction at unpaired bases and the unwinding of superhelical DNA with chemical probes.

Superhelical PM2 DNA I can be modified with N-cyclohexyl-N'-beta-(4-methylmorpholinium)ethyl carbodiimide (CMC). The transition of the sedimentation coefficient uncorrected for buoyant density change (S20,*) vs. % reactivity in terms of base pairs shows the following characteristics. The S20,* increases by 4.5 S units upon 1% modification. There is a plateau in S20,* between 1 and 4% reactivity. The extent of reactivity was determined by buoyant density and 14C radioactive CMC binding measurements. Further reactivity was not explored since Pulleyblank and Morgan's (22) data of S20,* vs. % reactivity from 6 to 34% was previously published. The initial results obtained in this study are complementary to the cited results of the above authors. Consequently, both sets of data taken together represent a complete description of S20,* vs. % reactivity with CMC. It is shown that the model in which superhelical DNA is proposed to contain small intrastrand hairpin regions can be extended to account for the observed transitions in S20,* vs. reactivity.

Carbodiimides↗

PubMed1997

[Effects of ethylene oxide on activity of DNA methylase in rats].

Male Wistar rats were exposed to ethylene Oxide (Eto) via inhalation at a concentration of 751.7 mg/m3 two hours a day and six days a week for nine consecutive weeks. Sensitivity test of restriction enzymes Mbo I, Sau3A I, Hpa II and Msp I and Southern blot were conducted to study the effects of ethylene oxide on activity of major intracellular modification enzyme, DNA methylase. Results showed that DNA in liver, kidney and testis of rats were completely enzymolysed and only one blot was seen after being treated with Hind III, Mbo I and Sau3A I, and incompletely enzymolysed and multiple blots were seen after being treated with Hpa II and Msp I. There were no significant differences in electrophoresis pattern, number of blots and intensity of radioautograph between the exposed animals and controls. It indicated that no effects of Eto on activity of DNA methylase in the liver, kidney and testis of rats was found, and degree of methylation in the testis was much less than that in the liver and kidney.

Animals↗

PubMed1994

Cloning and analysis of translational control for genes encoding the Cfr9I restriction-modification system.

The complete type-II Cfr9I restriction-modification (R-M) system of Citrobacter freundii strain RFL9, recognizing the DNA sequence CCCGGG, has been cloned and expressed, and functionally active enzymes have been produced in Escherichia coli. Both the methyltransferase (MTase; M.Cfr9I) and restriction endonuclease (ENase; R.Cfr9I) were found to be encoded on a 2.3-kb cloned fragment in the same transcriptional orientation, but differing in translational phases. The last codon (underlined) (ATGA) of the MTase-encoding gene (Cfr9IM) overlaps with the start codon for the ENase-encoding gene (overlined) (cfr9IR). A nucleotide sequence complementary to a predicted Shine-Dalgarno sequence preceding cfr9IR is within this gene. Predicted free energy (delta G) for formation of the mRNA secondary structure involving these complementary sequences was found to be -16.1 kcal/mol. Amino-acid sequence homology of 80% was found between R.Cfr9I and R.XcyI.

Amino Acid Sequence↗

PubMed2002

RAD6-dependent DNA repair is linked to modification of PCNA by ubiquitin and SUMO.

The RAD6 pathway is central to post-replicative DNA repair in eukaryotic cells; however, the machinery and its regulation remain poorly understood. Two principal elements of this pathway are the ubiquitin-conjugating enzymes RAD6 and the MMS2-UBC13 heterodimer, which are recruited to chromatin by the RING-finger proteins RAD18 and RAD5, respectively. Here we show that UBC9, a small ubiquitin-related modifier (SUMO)-conjugating enzyme, is also affiliated with this pathway and that proliferating cell nuclear antigen (PCNA) -- a DNA-polymerase sliding clamp involved in DNA synthesis and repair -- is a substrate. PCNA is mono-ubiquitinated through RAD6 and RAD18, modified by lysine-63-linked multi-ubiquitination--which additionally requires MMS2, UBC13 and RAD5--and is conjugated to SUMO by UBC9. All three modifications affect the same lysine residue of PCNA, suggesting that they label PCNA for alternative functions. We demonstrate that these modifications differentially affect resistance to DNA damage, and that damage-induced PCNA ubiquitination is elementary for DNA repair and occurs at the same conserved residue in yeast and humans.

Cell Cycle↗

PubMed2006

Retinoic acid receptor beta2 is epigenetically silenced either by DNA methylation or repressive histone modifications at the promoter in cervical cancer cells.

To elucidate the silencing mechanism of retinoic acid receptor beta2 (RAR beta2) in cervical carcinogenesis, we investigated RAR beta2 expression and the status of both DNA methylation and histone modifications at the promoter in cervical cancer cell lines. RAR beta2 was frequently repressed in cancer cell lines and in primary cancers of the cervix. Although the majority of RAR beta2-negative cancers had methylated promoter, RAR beta2 was repressed with hypomethylated promoter in a substantial fraction of the cancers. The RAR beta2-negative cells with hypomethylated promoters showed a repressive histone modification pattern at the promoter. RAR beta2 was reactivated by a histone deacetylase inhibitor, accompanied by formation of active histone modifications. The repressive modification was also observed in cells repressed with hypermethylated promoter, but RAR beta2 was reactivated only by DNA demethylating agent and not by histone deacetylase inhibitor. Our results suggest that RAR beta2 is silenced by either of the two key epigenetic pathways, DNA methylation or repressive histone modifications, depending on the individual cancer cells.

Base Sequence↗

PubMed2003

Cellular transformation of the investigational new anticancer drug NB1011, a phosphoramidate of 5-(2-bromovinyl)-2'-deoxyuridine, results in modification of cellular proteins not DNA.

NB1011 [E-5-(2-bromovinyl)-2'-deoxyuridine-5'-(L-methylalaninyl)-phenylphosphoramidate], a phosphoramidate prodrug of E-5-(2-bromovinyl)-2'-deoxyuridine-5'-monophosphate (BVdUMP), is an investigational new anticancer drug. NB1011 targets thymidylate synthase (TS), which catalyzes the transformation of BVdUMP into cytotoxic reaction products. Due to the elevated levels of TS expression in tumor cells compared to normal cells, these cytotoxic products are preferentially generated inside tumor cells, and, as expected, NB1011 is more toxic to cells with higher levels of TS expression. Therefore, NB1011 therapy should kill tumor cells without severely damaging normal cells. Radiolabeled NB1011 was used to determine the intracellular fate of NB1011 reaction products and, possibly, the mechanism of action of this investigational new drug. We found significant incorporation of the radiolabel into cellular macromolecules. In contrast to our expectations that NB1011 product(s) would be incorporated into DNA, we discovered that cellular proteins were the labeled macromolecular fraction. Herein, we report that the intracellular transformation of NB1011 involves formation of the corresponding monophosphate, TS-dependent transformation into highly reactive intermediates, and subsequent incorporation into cellular proteins. TS itself appears to escape irreversible inactivation. Our data suggest that protein modification not DNA incorporation accounts for the therapeutic effect of NB1011. The proposed mechanism is rather unexpected for a nucleotide analogue and could lead to the discovery of new cellular protein targets for future drug design.

Antineoplastic Agents↗

PubMed1992

Crystal and molecular structure of a DNA fragment containing a 2-aminoadenine modification: the relationship between conformation, packing, and hydration in Z-DNA hexamers.

The crystal and molecular structure of d(CGUA'CG)2 (where A' is 2-aminoadenine) has been determined and refined to an R factor of 13.8% for data 8.0-1.3 A. The structure is very similar to the original Z-DNA structures with the sequence d(CGCGCG)2 [Gessner, R. V., Frederick, C. A., Quigley, G. J., Rich, A., & Wang, A. H.-J. (1989) J. Biol. Chem. 264, 7921] and shows that the substitution of 2-aminoadenine-uracil base pairs in the two central steps is consistent with Z-DNA formation. In addition, we show how waters mediating intermolecular interactions may help to explain the ZI-ZII conformational pattern found in many Z-DNA structures.

2-Aminopurine↗

PubMed2003

Assembly of EcoKI DNA methyltransferase requires the C-terminal region of the HsdM modification subunit.

The methyltransferase component of type I DNA restriction and modification systems comprises three subunits, one DNA sequence specificity subunit and two DNA modification subunits. Limited proteolysis of the EcoKI methyltransferase shows that a 55-kDa N-terminal fragment of the 59-kDa modification subunit is resistant to degradation. We have purified this fragment and determined by mass spectrometry that proteolysis removes 43 or 44 amino acids from the C-terminus. The fragment fails to interact with the other subunits even though it still possesses secondary and tertiary structure and the ability to bind the S-adenosylmethionine cofactor. We conclude that the C-terminal region of the modification subunit of EcoKI is essential for the assembly of the EcoKI methyltransferase.

Amino Acid Sequence↗

PubMed1998

Binding of human anti-DNA autoantibodies to reactive oxygen species modified-DNA and probing oxidative DNA damage in cancer using monoclonal antibody.

The binding of native and reactive oxygen species-modified DNA (ROS-DNA) to circulating antibodies in the serum of patients with various types of cancer has been investigated by competition enzyme-linked immunosorbent assay. Fifteen sera of 35 showed reactivity with native and/or ROS-DNA. Eleven of these showed higher binding to ROS-DNA (36-64% inhibition), whereas 1 showed higher reactivity with native DNA (nDNA) (42% inhibition). Three sera reacted with both native and ROS-DNA almost equally. Oxidative lesions in human genomic DNA were immunochemically detected using an anti-ROS-DNA monoclonal antibody (MAb) probe. Two of 3 DNA isolates from blood of breast cancer patients, 1 of 3 from lung cancer and 1 of 2 each from hepatocellular cancer and cancer of the gallbladder were reactive with the MAb. Higher recognition of ROS-DNA by circulating antibodies and DNA isolated from cancer patients by the MAb indicates increased oxidative stress leading to DNA damage. Our results suggest that ROS modification of DNA probably alters its immunogenicity leading to the generation of antibodies to ROS-DNA, probably by the activation of autoreactive cells. The induced antibodies against modified DNA are cross-reactive to native DNA.

Adult↗

PubMed1998

News & notes. Efficient library construction with a TA vector and its application to cloning of the phytoene synthase gene from the cyanobacterium Spirulina platensis.

An efficient and simple method for constructing a genomic DNA library is presented by use of a TA cloning vector. It is based on sonicative cleavage of genomic DNA and modification of the fragment ends with Taq DNA polymerase, followed by ligation with a TA vector. This method was successfully applied to cloning of the phytoene synthase gene crtB from Spirulina platensis. The method is useful when the genomic DNA is not well digested with restriction enzymes owing to methylation or other reasons.

Alkyl and Aryl Transferases↗

PubMed1996

The effect of ionic strength on melting of DNA modified by platinum(II) complexes.

Thermal denaturation of calf thymus DNA modified by antitumor cis-diamminedichloroplatinum(II) (cis-DDP) and by two related Pt(II) compounds which had been shown to be clinically ineffective, viz. trans-diamminedichloroplatinum(II) (trans-DDP) or monodentate diethylenetriaminechloroplatinum(II) chloride [[Pt(dien)Cl)]Cl], was studied by monitoring changes of absorbance at 260 nm. The melting of DNA platinated to different levels was investigated in neutral media containing varying concentrations of Na+. It has been shown that the ionic strength has a strong influence on the character and magnitude of changes in the melting temperature of DNA (Tm) induced by the platination. The modification of DNA by either platinum complex used in this work results in an increase of Tm if DNA melting is measured in media containing low Na+ concentrations (ca. 1 mM). This effect is reversed at higher Na+ concentrations. The concentration of Na+ at which this reversal occurs is, however, markedly lower for DNA modified by cis-DDP than for DNA modified by the other two platinum complexes. These results have been interpreted to mean that at least three factors affect the thermal stability of DNA modified by the platinum(II) complexes: stabilization effects of the positive charge on the platinum moiety and of interstand cross-links, and a destabilization effect of conformational distortions in DNA. Thus, in order to compare and interpret the melting behavior of DNA modified by different compounds, a great attention has to be paid to the composition of the medium in which the melting experiments are carried out.

Animals↗

PubMed2001

DNA methylation: an alternative pathway to cancer.

OBJECTIVE: To provide an introduction to the concept of DNA methylation and its function in normal cells, and to explain the possible mechanisms as to how abnormalities in this phenomenon can relate to carcinogenesis. The clinical implications with reference to common malignancies encountered in surgical practice are discussed. SUMMARY BACKGROUND DATA: Methylation of DNA is a heritable, enzyme-induced modification to DNA structure without alteration of the specific sequence of the base pairs responsible for encoding the genome. DNA methylation can both directly inhibit the expression of genes and also increase the probability that affected genes undergo a mutational event. Although DNA methylation plays an essential role in normal biologic processes, distinct and abnormal patterns of methylation are observed in cancers. In particular, there has been increased documentation that methylation of the promoter regions of several genes, including known tumor suppressor genes, results in the subsequent failure to express their functional proteins. Consequently, DNA methylation may represent an early and fundamental step in the pathway by which normal tissue undergoes neoplastic transformation. Further, an assessment of the methylation profiles within neoplastic tissues may provide key information in enhancing the diagnosis, predicting the clinical behavior, and designing specific treatment plans for individual patients. METHODS: Published literature from 1925 to 2000 contributing to an understanding of the purpose of DNA methylation and how pathology of this phenomenon could contribute to cancer are reviewed. Theories on these issues and the evidence that led to them are described. The present status of the subject in a clinical context is discussed. RESULTS: Gene expression can be significantly modulated by alterations in DNA methylation patterns. Methylation within the promoter regions of tumor suppressor genes causes their silencing, and methylation within the gene itself can induce mutational events. These mechanisms may play a fundamental role in precipitating the development of a large and diverse number of human cancers. CONCLUSIONS: DNA methylation is an important factor in the development of cancer. A greater understanding of the relationship between DNA methylation events at the molecular level and its interaction in the clinical context may provide the basis for future advances in the surgical and pharmacologic management of malignant diseases.

Breast Neoplasms↗

PubMed1994

Active maize genes are unmodified and flanked by diverse classes of modified, highly repetitive DNA.

We have characterized the copy number, organization, and genomic modification of DNA sequences within and flanking several maize genes. We found that highly repetitive DNA sequences were tightly linked to most of these genes. The highly repetitive sequences were not found within the coding regions but could be found within 6 kb either 3' or 5' to the structural genes. These highly repetitive regions were each composed of unique combinations of different short repetitive sequences. Highly repetitive DNA blocks were not interrupted by any detected single copy DNA. The 13 classes of highly repetitive DNA identified were found to vary little between diverse Zea isolates. The level of DNA methylation in and near these genes was determined by scoring the digestibility of 63 recognition/cleavage sites with restriction enzymes that were sensitive to 5-methylation of cytosines in the sequences 5'-CG-3' and 5'-CNG-3'. All but four of these sites were digestible in chromosomal DNA. The four undigested sites were localized to extragenic DNA within or near highly repetitive DNA, while the other 59 sites were in low copy number DNAs. Pulsed field gel analysis indicated that the majority of cytosine modified tracts range from 20 to 200 kb in size. Single copy sequences hybridized to the unmodified domains, while highly repetitive sequences hybridized to the modified regions. Middle repetitive sequences were found in both domains.

Chromosome Mapping↗

PubMed1992

[Detection of left-helical segments in eukaryotic DNA].

The method of DNA binding to nitrocellulose filters was applied to DNA isolated from mouse liver and Ehrlich ascite carcinoma (EAC), calf thymus, and lymphocytes from patients with chronic lymphoid leukemia. In those and phage PM2 DNA the increase in the DNA binding to the filters with a rise in NaCl concentration from 0.5 up to 4.5 M was sigmoidal being suggestive of a conformational transition. No such activity was found in the case of phage lambda or single-stranded DNA. The binding decreased dramatically after mild cleavage of DNA with DNAase I or treatment with phospholipase C or Eco RI and Hin PI restrictases. Incubation of DNA with ethidium bromide led to decrease in the amount of bound DNA. This effect was enhanced with a rise in the dye concentration. The isotherms of ethidium bromide binding to eukaryotic DNA obtained in Scatchard plots by optic titration had a component with a positive slope at low values of r. Bivalent ions (Mg2+, Zn2+) shifting the equilibrium towards the Z-form increased the proportion of macromolecules retained on the filters at NaCl concentrations of 1-3 M. Local changes in the helix conformation were studied with the help of chemical probes: diethylpyrocarbonate (guanine Z-DNA) and osmium-pyridine reagent (pyrimidines of boundary B-Z sites). These probes incorporation into samples of liver DNA, EAC, and lymphocytes resulted in chemical modification of all these samples. Modification of DNA by osmium-pyridine reagent led to inhibition of subsequent restriction by Eco RI restrictase. The data obtained are suggestive of the presence of Z-regions in the B-helix of eukaryotic DNA. A topological model of Z-site stabilization in small superhelical loops of DNA fixed by protein or lipoprotein molecules is proposed.

Animals↗