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Biomedical subjects

M Leng

Publications and source records attributed to M Leng.

At least 91 records · Page 5Linked to original sources
PubMed1988

N-acetoxy-N-2-acetylaminofluorene-induced damage on SV40 DNA: inhibition of DNA replication and visualization of DNA lesions.

The chemical carcinogen, N-acetoxy-N-2-acetylaminofluorene (AAAF), which induces well characterized DNA lesions, strongly inhibits Simian virus 40 (SV40) DNA replication. By using SV40 mutants which were temperature-sensitive for replication initiation, we were able to synchronize SV40 DNA replication and therefore to introduce AAAF-induced lesions only on unreplicating SV40 molecules. One to two acetylaminofluorene (AAF)-adducts per SV40 genome inhibit more than 90% of normal semi-conservative DNA synthesis. SV40 replicative intermediates (RIs) from AAAF-treated infected cells, purified through neutral sucrose gradients and BND-cellulose column, possess a structure different from the usual Cairns molecules found in the untreated cultures. Both by neutral and alkaline sucrose gradients and by electron microscopy, the RIs isolated from treated cells appear as complex molecules with single-stranded portions and sometimes with a tailed structure. Moreover, the newly synthesized strands found in these molecules are equal in size to the average distance between AAF-adducts on the template strand, indicating that AAF-adducts represent a block for the SV40 DNA replication. By using specific anti Guo-AAF antibodies and electron microscopy, we show the presence of an AAF adduct at halted replication forks, i.e. showing a DNA replication block in a mammalian replicon for the first time. We therefore assume that AAF-adducts severely block the progression of the replication forks by inhibiting, at least, the in vivo polymerization of the leading strand.

2-Acetylaminofluorene↗
PubMed1987

Slow exchanging protons in the Z-form of G-C and A-C alternating polymers by using a rapid dialysis method.

Using a dialysis method we have measured the hydrogen exchange (HX) kinetics in poly(dG-dC).poly(dG-dC), poly(dG-m5dC).poly(dG-m5dC), poly(dG-br5dC).poly(dG-br5dC) and platinated poly(dA-br5dC).poly(dG-dT) under experimental conditions in which these polymers adopt the Z-conformation. The latter polymer has one slow exchanging proton with a half-time of about 2 h, whereas the other G-C alternating polymers display a slow class of two protons with exchange half-time of about 6 h. These exchange half-times are independent of ionic strength and of the nature of the salt for all these polymers in the Z-form. The slow proton exchange appears to be strongly correlated to the Z-conformation but rather independent of the Z-DNA sequence. The comparison of the proton exchange rates with the corresponding B in equilibrium Z transition rates is not in favour of the same rate limiting step for both processes.

Circular Dichroism↗
PubMed1987

Reaction of nucleic acids with cis-diamminedichloroplatinum(II): interstrand cross-links.

In the reaction of cis-diamminedichloroplatinum(II) (cis-DDP) with double-helical (dC-dG)4.(dC-dG)4 or (dC-dG)5.(dC-dG)5, intrastrand and interstrand cross-links between two guanine residues are formed. This is shown by gel electrophoresis in denaturing conditions of the reaction products and by high-performance liquid chromatography (HPLC) analysis of the products digested with nuclease P1. In the reaction of cis-DDP and poly(dG-dC).poly(dG-dC), at relatively low levels of platination, it is mainly interstrand cross-links between two guanine residues that are formed. This is shown by HPLC analysis of the nuclease P1 digest and by gel electrophoresis in denaturing and nondenaturing conditions of the platinated polymer after cleavage with the restriction enzyme HhaI. Moreover, the antibodies to platinated poly(dG-dC).poly(dG-dC) cross-react with the interstrand cross-linked (dC-dG)4 or (dC-dG)5 but not with the intrastrand cross-linked (dC-dG)4 or (dC-dG)5. These antibodies cross-react with platinated natural DNA. The amount of interstrand cross-links deduced from radioimmunoassays (0.5% of the total bound platinum) is lower than that (2%) deduced by gel electrophoresis in denaturing conditions of a platinated DNA restriction fragment. By gel electrophoresis, it is also shown that in vitro the isomer trans-DDP is more efficient in forming interstrand cross-links than cis-DDP.

Antibodies↗
PubMed1987

Comparison of the reactivity of B-DNA and Z-DNA with two isosteric chemical carcinogens: 2-N,N-acetoxyacetylaminofluorene and 3-N,N-acetoxyacetylamino-4,6-dimethyldipyrido-[1,2-a:3',2' -d] imidazole.

The reactivity of nucleic acids in various conformations and two isosteric chemical carcinogens 2-N,N-acetoxyacetylaminofluorene (N-AcO-AAF) and 3-N,N-acetoxyacetylamino-4,6-dimethyldipyrido [1,2-a:3',2'-d] imidazole (N-AcO-AGlu-P-3) have been studied. Both carcinogens bind covalently to poly(dG-dC).poly(dG-dC) (B form) and to poly(dG-br5C).poly(dG-br5dC) (Z form). They also bind covalently to (dC-dG)16 and to (dG-dT)15 sequences inserted in plasmids when the inserts are in the B form but they do not bind to the inserts in the Z form. The reactivity of guanine residues at the B-Z junctions depends upon the superhelical density of the plasmids and upon the base sequences at the junction. The distribution of AGlu-P-3 modified guanines in a restriction fragment of pBR322 is not uniform and is different from that of AAF-modified guanines. The conclusion is that N-AcO-Glu-P-3 as N-AcO-AAF can probe at the nucleotide level the polymorphism of DNA. On the other hand, the non-reactivity of both chemical carcinogens and Z-DNA and the hyperreactivity of some junctions might have some importance in the understanding of chemical carcinogenesis.

2-Acetylaminofluorene↗
PubMed1987

Characterization of the ternary complexes formed in the reaction of cis-diamminedichloroplatinum (II), ethidium bromide and nucleic acids.

The purpose of this study was to characterize the ternary complexes formed in the reaction of cis-diamminedichloroplatinum (II) (cis-DDP) and nucleic acids, in the presence of the intercalating compound ethidium bromide (EtBr). In these ternary complexes, some EtBr is tightly bound to the nucleic acids. Tight binding is defined by resistance to extraction with butanol, assayed by filtration at acid pH or thin layer chromatography at basic pH. These ternary complexes are formed with double stranded but not with single stranded nucleic acids. They are not formed if cis-DDP is replaced by transdiamminedichloroplatinum(II). The amount of tightly bound EtBr depends upon the sequence of the nucleic acid, being larger with poly (dG-dC).poly(dG-dC) than with poly(dG).poly(dC). Spectroscopic results support the hypothesis that the tight binding of the dye is due to the formation of a bidentate adduct (guanine-EtBr)cis-platin. The visible spectrum of the ternary complexes is blue-shifted as compared to that of EtBr intercalated between the base pairs of unplatinated DNA and it depends upon the conformation of the ternary complex. The fluorescence quantum yield of the ternary complexes is lower than that of free EtBr in water. Tightly bound EtBr stabilizes strongly the B form versus the Z form of the ternary complex poly(dG-dC)-Pt-EtBr and slows down the transition from the B form towards the Z form. The sequence specificity of cis-DDP binding to a DNA restriction fragment in the absence or presence of EtBr is mapped by means of the 3'----5' exonuclease activity of T4 DNA polymerase. In the absence of the dye, all the d(GpG) sites and all the d(ApG) sites but one in the sequence d(TpGpApGpC) are platinated. The d(GpA) sites are not platinated. In the presence of EtBr, some new sites are detected. These results might help to explain the synergism for drugs used in combination with cis-DDP and in the design of new chemotherapeutic agents.

Chemical Phenomena↗
PubMed1986

N-hydroxyaminofluorene: a chemical probe for DNA conformation.

The importance of the polymorphism of DNA in the reaction with the chemical carcinogen N-hydroxyaminofluorene is studied by means of a supercoiled plasmid containing an insert of (dC-dG). Immunochemical titration and the determination of the binding spectrum of -aminofluorene adducts show that the carcinogen reacts with B-DNA but not with Z-DNA and that conformational changes of the B-DNA-Z-DNA junctions occur as a function of the superhelical density.

Carcinogens↗
PubMed1986

Poly(dA-dT).poly(dA-dT) two-pathway proton exchange mechanism. Effect of general and specific base catalysis on deuteration rates.

The deuteration rates of the poly(dA-dT).poly(dA-dT) amino and imino protons have been measured with stopped-flow spectrophotometry as a function of general and specific base catalyst concentration. Two proton exchange classes are found with time constants differing by a factor of 10 (4 and 0.4 s-1). The slower class represents the exchange of the adenine amino protons whereas the proton of the faster class has been assigned to the thymine imino proton. The exchange rates of these two classes of protons are independent of general and specific base catalyst concentration. This very characteristic behavior demonstrates that in our experimental conditions the exchange rates of the imino and amino protons in poly(dA-dT).poly(dA-dT) are limited by two different conformational fluctuations. We present a three-state exchange mechanism accounting for our experimental results.

Deuterium↗
PubMed1986

Nucleotide sequence of an heterochromatic segment recognized by the antibodies to Z-DNA in fixed metaphase chromosomes.

The purpose of this work was to analyse at the molecular level the DNA recognized by the antibodies to Z-DNA in in situ experiments. Antibodies to Z-DNA interact strongly with R-band positive heterochromatic segments of fixed metaphase chromosomes of Cebus (Viegas-Pequignot et al., 1983). These segments are constituted of a satellite DNA the repeat unit of which is about 1520 base pairs long. The base sequence of the repeat unit has been determined. It contains a (AC)n rich region which, in vitro, adopts the Z conformation under topological constraints. Experiments with nuclei suggest that this sequence is not predominantly in the Z conformation in vivo. The polymorphic structure of the (AC)n rich region argues for an active recombination sequence.

Animals↗
PubMed1986

A study of the thermal stability of the Z form of (m5dC-dG)3 by resonance Raman spectroscopy.

The thermal stability of the hexanucleoside pentaphosphate m5dCpdGpm5dCpdGpm5 dCpdG has been studied by resonance Raman spectroscopy with 257 nm excitation wavelength. At low temperature and in 3M NaClO4, the Raman spectrum resembles that of poly(dG-dC).poly(dG-dC) in the Z conformation. As the temperature is increased, the position and the intensity of several bands (1312 cm-1, 1482 cm-1, 1584 cm-1 and 1632 cm-1) are modified. The variation of intensity versus temperature is biphasic. Analysis of the results suggests that the increase of temperature induces first a transition from the Z form to an intermediate stable form which then melts. These results and those previously obtained by circular dichroism and 31P nuclear magnetic resonance suggest that the intermediate form belongs to the left family but with changes in the stacking of the bases and the geometry of the phosphate groups as compared to the canonical Z form.

Circular Dichroism↗
PubMed1986

Solvation of the left-handed hexamer d(5BrC-G-5BrC-G-5 BrC-G) in crystals grown at two temperatures.

Crystals of the hexadeoxyoligomer d(5BrC-G-5BrC-G-5BrC-G) were grown at different temperatures (5 degrees C, 18 degrees C and 37 degrees C) in the absence of divalent cations. The crystals grown at 5 degrees C did not diffract X-rays, while those grown at 18 degrees C and 37 degrees C did. The oligomer adopts the left-handed ZI conformation in both crystals. The main difference resides in a more extensive hydration shell in the crystal grown at high temperature than in the crystal grown at low temperature. The high-temperature crystal displays a spine of hydration running deep in the minor groove and linking exocyclic O-2 atoms of the pyrimidine rings. In both crystalline forms, a hydrated sodium ion bound to the N-7 of a guanine ring was found. Strings of water molecules bridging phosphate anionic oxygen atoms are found along the backbone. The absolute values of the propeller-twist are also different in both structures although the values of the twist are very similar. The results point to the importance of the crystallization conditions when analysing fine structural details like solvation properties of oligomers.

Crystallization↗
PubMed1986

In vitro enzymatic methylation of DNA modified with the mutagenic amine: 3-N,N-acetoxyacetylamino-4,6-dimethyldipyrido(1,2-a:3'2'-d )imidazole.

Both the initial velocity and the overall methylation of DNA modified by acetylamino-4,6-dimethyldipyrido(1,2-a:3',2'-d)imidazole (A-Glu-P-3) by rat liver DNA-(cytosine-5-)-methyltransferase are decreased as compared to native DNA. A-Glu-P-3 bound to guanine residues may block the movement of the enzyme along the helix. The modified DNA does not inhibit the enzymatic methylation of native DNA. The enzyme has a lower affinity for the modified DNA than for native DNA. The hypomethylation caused by this carcinogen could have a significance in gene activity, cellular differentiation and cancer induction.

2-Acetylaminofluorene↗
PubMed1986

Thermal stability of the Z conformation of the hexanucleoside pentaphosphate d(br5CGbr5CGbr5CG): evidence for a conformational transition before melting.

The thermal stability of the hexanucleoside pentaphosphate d(br5CGbr5CGbr5CG) has been studied at two nucleotide concentrations, in the presence of 1 M NaClO4. At low nucleotide concentration (7 X 10(-5) M), circular dichroism experiments show a conformational transition from the Z conformation to another conformation, named X, which is not the B conformation, as the temperature is increased from 0 to 35 degrees C. Between 40 and 65 degrees C, another transition is observed which corresponds to the melting of the X conformation. At higher nucleotide concentration (2 X 10(-3) M), circular dichroism and 31P nuclear magnetic resonance experiments show that at low temperature (br5dC-dG)3 adopts the Z conformation. There are associations between the oligonucleotides which progressively disappear as the temperature increases. In the range 35-60 degrees C a transition from the Z conformation to another conformation is observed. This new conformation is the X conformation detected at low nucleotide concentration.

Circular Dichroism↗
PubMed1986

Reaction of nucleic acids and cis-diamminedichloroplatinum(II) in the presence of intercalating agents.

The reaction of cis-diamminedichloroplatinum(II) and several synthetic or natural double-stranded polydeoxyribonucleotides has been carried out in the presence of such intercalating agents as ethidium bromide, proflavine, and acridine. After incubation of the reaction mixtures at 37 degrees C for 24 hr, some ethidium or proflavine, but no acridine, molecules are tightly bound to nucleic acids. Tight binding is defined by resistance to extraction with butanol, assayed by filtration at acid pH or by thin-layer chromatography at basic pH. In the ternary complexes, there is about one tightly bound ethidium (or proflavine) per platinum residue. At 37 degrees C, but not at 4 degrees C, tightly bound ethidium exchanges with free ethidium, whereas platinum residues do not exchange. The binding and the release of tightly bound ethidium are very slow (several hours). It is suggested that in the ternary complexes, nucleic acid-cis-Pt(NH3)2-intercalating agent, a bidentate adduct (guanine-ethidium or -proflavine)-cis-Pt(NH3)2, is formed. No tightly bound ethidium or proflavine is found when cis-diamminedichloroplatinum(II) is replaced by trans-diamminedichloroplatinum(II). Competition experiments between cis-diamminedichloroplatinum(II), poly(dG-dC), and poly(dG)-poly(dC) or poly(dA-dT) show that the presence of ethidium bromide, proflavine, or acridine interferes with the distribution of platinum between the polynucleotides. These results might help to explain the synergism for drugs used in combination with cis-diamminedichloroplatinum(II) and in the design of new chemotherapeutic agents.

Acridines↗
PubMed1986

In situ hybridization of an acetylaminofluorene-modified probe recognized by Z-DNA antibodies in vitro.

An in situ hybridization procedure, based on the chemical modification of DNA by acetylaminofluorene (AAF), followed by a specific immunoreaction was used to localize a Z-DNA sequence isolated from the satellite DNA of Cebus appella. The AAF probe is localized on the R-band-positive heterochromatic segments of Cebus chromosomes, which strongly react with Z-DNA antibodies. The use of a nonradioactive single-stranded labeled probe confirms the reliability and the rapidity of immunochemical methods for the detection of DNA sequences on chromosomes.

2-Acetylaminofluorene↗
PubMed1985

Left-handed Z-DNA.

Since the Watson-Crick proposal of right-handed B-DNA, numerous studies have been devoted to the conformation of DNA. Both natural DNAs of heterogeneous sequences and synthetic DNAs are capable of adopting more than one conformation. The specific conformation a DNA adopts appears to depend mainly on its base sequence and its environmental conditions. For a given DNA, changes in environmental conditions can induce conformational transitions which occur according to cooperative or non-cooperative processes (for general reviews see Ref. 1a, b). Despite many results, molecular biologists did not put much emphasis on the polymorphism of DNA. The discovery of the intraconversion in helical sense between the right-handed B and left-handed Z conformers of DNA has brought a new interest in the polymorphism of DNA. It is now proposed that this polymorphism has important functions in biological reactions. A recent review, 'The Chemistry and Biology of Left-handed Z-DNA', by Rich et al. has just been published. We here report some of the results published in 1984 on Z-DNA.

Animals↗
PubMed1985

"Asymmetric" opening reaction mechanism of Z-DNA base pairs: a hydrogen exchange study.

With the tritium-Sephadex method, the hydrogen-exchange kinetics of the five NH protons of guanine and cytosine residues in Z-form poly(dG-dC) X poly (dG-dC) were measured as a function of temperature and catalyst concentration. Over the measured temperature range from 0 to 34 degrees C, two classes of protons with constant amplitudes are found. The three protons of the fast class, which were assigned to the guanine amino and imino protons, have an exchange half-time in the minute time range (at 20 degrees C the half-time is 2.5 min) and an activation energy of 18 kcal mol-1. Since these two types of protons exchange at the same rate in spite of their grossly different pK values, the exchange of these protons must be limited by the same nucleic acid conformational change. The two cytosine amino protons of the slow class are especially slow with exchange half-times in the hour time range (at 20 degrees C the exchange half-time is 1 h) and the activation energy is 20 kcal mol-1. The exchange of these two protons is not limited by some nucleic acid conformational change as shown by the marked exchange acceleration of these protons upon addition of 0.2 M imidazole. In addition, we have also reexamined the hydrogen-deuterium exchange kinetics of the amino protons of guanosine cyclic 2',3'-monophosphate by a spectral difference method using a stopped-flow spectrophotometer. The measured kinetic process is monophasic with a rate constant of 3 s-1 at 20 degrees C, which is in the same range as the predicted rate constant of the guanine amino protons.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Composition↗
PubMed1985

Comparison between poly(dG-dC).poly(dG-dC) and DNA modified by cis-diamminedichloroplatinum (II): immunological and spectroscopic studies.

The importance of the base composition and of the conformation of nucleic acids in the reaction with the drug cis-diamminedichloroplatinum(II) has been studied by competition experiments between the drug and several double-stranded polydeoxyribonucleotides. Binding to poly(dG).poly(dC) is larger than to poly (dG-dC).poly(dG-dC). There is no preferential binding in the competition between poly(dG-dC).poly(dG-dC), poly(dA-dC).poly(dG-dT) and poly(dA-dG).poly(dC-dT). In the competition between poly(dG-dC).poly (dG-dC) (B conformation) and poly(dG-br5dC).poly(dG-br5dC) (Z conformation), the drug binds equally well to both polynucleotides. In natural DNA, modification of guanine residues in (GC)n.(GC)n sequences by the drug has been revealed by the inhibition of cleavage of these sequences by the restriction enzyme BssHII. By means of antibodies to platinated poly(dG-dC), it is shown that some of the adducts formed in platinated poly(dG-dC) are also formed in platinated pBR322 DNA. The type of adducts recognized the antibodies is not known. Thin layer chromatography of the products after chemical and enzymatic hydrolysis of platinated poly(dG-dC) suggests that interstrand cross-links are formed. Finally, the conformations of poly(dG-dC) modified either by cis-diamminedichloroplatinum(II) or by trans-diamminedichloroplatinum (II) have been compared by circular dichroism. Both the cis-isomer and the trans-isomer stabilize the Z conformation when they bind to poly(dG-m5dC) in the Z conformation. When they bind to poly(dG-m5dC) in the B conformation, the conformations of poly(dG-m5dC) modified by the cis or the trans-isomer are different. Moreover, the cis-isomer facilitates the B form-Z form transition of the unplatinated regions while the trans-isomer makes it more difficult.

Binding, Competitive↗