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A Mazen

Publications and source records attributed to A Mazen.

At least 19 recordsLinked to original sources
PubMed1989

Poly(ADP-ribose)polymerase: a novel finger protein.

By Energy Dispersive X-ray fluorescence we have determined that calf thymus poly(ADP-ribose) polymerase binds two zinc ions per enzyme molecule. Using 65Zn (II) for detection of zinc binding proteins and polypeptides on western blots, we found that the zinc binding sites are localized in a 29 kd N-terminal fragment which is included in the DNA binding domain. Metal depletion and restoration experiments proved that zinc is essential for the binding of this fragment to DNA as tested by Southwestern assay. These results correlate with the existence of two putative zinc finger motifs present in the N-terminal part of the human enzyme. Poly(ADP-ribose)polymerase fingers could be involved in the recognition of DNA strand breaks and therefore in enzyme activation.

Amino Acid Sequence↗
PubMed1988

Structural and functional analysis of poly(ADP ribose) polymerase: an immunological study.

Poly(ADP ribose) polymerase (EC 2.4.2.30) was studied using monoclonal antibodies for three different epitopes on the enzyme. The epitopes were mapped in relation to the functional domains of the protein and the inhibitory properties of the antibodies. The intranuclear and interspecies immunoreactivity of the enzyme was also investigated. The epitope of antibody 2 was mapped to the 17 kDa fragment generated by chymotryptic digestion of the C-terminal 54 kDa NAD-binding domain. Antibody 9 binds to the N-terminal 29 kDa fragment of the DNA binding domain and inhibits the enzyme activity by 80%. This antibody was used to purify poly(ADP ribose) polymerase by immunoaffinity chromatography. The third antibody binds to a central 36 kDa fragment that possesses part of the DNA-binding domain and the automodification domain. This antibody increases the enzymatic activity by 30%. An analysis of the species cross-reactivity of the antibodies was carried out by immunoblot analysis of nuclear proteins. Antibody 10 binding was detected in rat FR3T3 cells, Chinese hamster ovary cells (CHO) and epidermoid carcinoma lung human cells (CALU-1). The other two antibodies are specific for the human and bovine enzymes. Western blot analysis showed the association of poly(ADP ribose) polymerase with residual nuclear material obtained after nuclease treatment and high-salt extraction. Immunofluorescence studies with the three different monoclonals demonstrated that accessibility of the epitopes varies in the nucleus.

Animals↗
PubMed1988

Zinc-binding proteins detected by protein blotting.

The Western blotting technique was used for the detection of zinc-binding proteins. Proteins were separated electrophoretically on 15% polyacrylamide-sodium dodecyl sulfate minigels, the gels were soaked in a reduction buffer, and the proteins were transferred to nitrocellulose filters. Zinc-binding proteins were probed with radioactive zinc (65Zn) and were detected by autoradiography. This technique allows the detection of as little as 20 to 100 pmol of zinc metalloproteins.

Autoradiography↗
PubMed1987

Poly(ADP-ribose) polymerase forms loops with DNA.

The interaction between highly purified poly(ADP-ribose) polymerase from calf thymus and different topological forms of pBR322 DNA has been studied by gel retardation electrophoresis and electron microscopy. We show that: (i) in the absence of nicks on DNA the enzyme has a marked affinity for supercoiled (form I) DNA, (ii) in the presence of single stranded breaks poly(ADP-ribose) polymerase preferentially binds to form II, (iii) in all cases enzyme molecules are frequently located at DNA intersections, (iv) a cooperative binding of the enzyme on DNA occurs.

DNA↗
PubMed1987

H3 phosphorylation-dependent structural changes in chromatin. Implications for the role of very lysine-rich histones.

Contrary to native H1/H5-containing chromatin where phosphorylation induces local structural changes affecting chromatin condensation, in stripped fibers phosphorylation of the totality of H3 molecules does not affect significantly chromatin conformation and DNA-protein interactions. Modification of H3 causes only a slight increase of flexibility of nucleosomal chains, despite important changes in histone topography revealed by immunochemical reactivity studies. We suggest that phosphorylation may only induce into the system the potential for dynamic change by modulating histone-histone interactions within and between nucleosomes, probably as a result of conformational change in the H3 protein. The signal for structural change would come from one or other factors (very lysine-rich histones, non-histones) that influence internucleosomal interactions at very specific locations in the chromatin, probably through protein-protein contacts. So, phosphorylation may modify a direct interaction between the N-terminal basic tail of H3 and very lysine-rich histones.

Birefringence↗
PubMed1985

Histone phosphorylation in native chromatin induces local structural changes as probed by electric birefringence.

In order to understand how the phosphorylation of histones affects the chromatin structure, we used electron microscopy, sedimentation velocity, circular dichroism and electric birefringence to monitor the salt-induced filament reversible solenoid transition of phosphorylated and native chromatin. Phosphorylation in vitro of chicken erythrocyte chromatin by cyclic-AMP-dependent protein kinase from porcine heart led to the modification of the histones H3 and H5 only, which were modified at a level of one phosphate and about three phosphate groups per molecule, respectively. In contrast to circular dichroism and sedimentation studies, which tend to suggest that phosphorylation of H3 and H5 does not affect chromatin structure, electron microscopy reveals that phosphorylation causes a relaxation of structure at low ionic strength. Electric birefringence and relaxation time measurements clearly prove that local structural changes are induced in chromatin: we observe a decrease of the steady-state birefringence with the appearance of a negative contribution in the signal and a marked increase of the flexibility of fibres. The component with the negative birefringence presents very short relaxation times, like those exhibited by small DNA fragments or individual nucleosomes. Two possibilities are then suggested. First, the conformational change is consistent with what would be expected from the presence of DNA segments loosely associated with the core histone H3. That the length of such segments could correspond to about one to two base-pairs per nucleosome strongly suggests that phosphorylation induces changes affecting some specific H3-DNA interactions only. This result could corroborate previous observations indicating that the N-terminal region of H3, where the site of phosphorylation is located, plays a decisive role in maintaining the superstructure of chromatin. Second, phosphorylation could introduce hinge points between each nucleosome. In this case, the negative birefringence results from partial orientation of the swinging nucleosomes. A possible mode of action of phosphorylation might be to weaken structural restraints imposed by histone H3, thus facilitating further condensation of chromatin.

Animals↗
PubMed1984

Use of histone antibodies for studying chromatin topography and the phosphorylation of chromatin subunits.

Polyclonal and monoclonal antibodies specific for histones as well as sera directed against synthetic peptides of histones were used to probe the topography of chromatin subunits. In native chromatin, the regions corresponding to residues 130-135 of H3 and 6-18 of H2B were found to be exposed and able to interact with antibodies whereas the regions 26-35 and 36-43 of H2B and 80-89 and 85-102 of H4 were not. In vitro phosphorylation of H3 and H5 in native chromatin or of H3 in H1/H5-depleted chromatin led to a marked drop in the binding of antibodies specific for residues 130-135 of H3 and 6-18 of H2B. Phosphorylation of H1/H5-depleted chromatin also altered the degree of exposure of certain H2A epitopes but it did not affect the surface accessibility of residues 1-11 of H2B.

Animals↗
PubMed1982

Non-random binding of N-acetoxy-N-2-acetylamino-fluorene to chromatin subunits as visualized by immunoelectron microscopy.

The influence of chromatin structure on the accessibility of DNA to the model ultimate carcinogen N-acetoxy-N-2-acetylaminofluorene (N-Aco-AAF) was investigated by means of an immunoelectron microscopic technique developed recently. An homogeneous population of core particles or trinucleosomes from chicken erythrocytes, was submitted to electrophilic attack by N-Aco-AAF. After DNA isolation, N-2-acetylaminofluorene (AAF) binding sites were mapped upon the DNA fragments using specific antibodies as a probe. Our results indicate a non-random binding of AAF along the DNA. Our data support the results of previous studies showing a preferential binding on the linker region.

2-Acetylaminofluorene↗
PubMed1982

Localization of histone H5 in the subunit organization of chromatin using immunoelectron microscopy.

In avian erythroid cells the erythrocyte-specific histone H5 is involved, like H1, in the packing of nucleosomes in the 25-nm chromatin fibers. In this study the distribution of histone H5 along the polynucleosomal chains was visualized by immunoelectron microscopy. Trinucleosomes from chicken erythrocytes and liver were used in order to test the specificity of the reaction with purified rabbit anti-H5 antibodies at various ionic strengths (5-80 mM). Long-chain chromatin was then reacted with anti-H5 antibodies and with sorted monomeric ferritin conjugate under chosen conditions. The antigenic determinants of histone H5 in the 25-nm fiber of long-chain chromatin (at 80 mM NaCl) are as accessible to the specific antibodies as in trinucleosomes. When the immunocomplexes were examined by electron microscopy in a low-ionic-strength buffer, permitting maximum extension of the chromatin structure on the grid, clusters of compacted nucleosomes were seen, separated by short regions of relaxed nucleosomes. Single nucleosomes enlarged by the antibodies are sometimes visible in the extended domains. We conclude that histone H5 is located primarily on series of adjacent nucleosomes but it can also be found on single nucleosomes located in the H1-enriched extended domains.

Animals↗
PubMed1982

Adenosine diphosphate ribosylation of chicken-erythrocyte histones H1, H5 and high-mobility-group proteins by purified calf-thymus poly(adenosinediphosphate-ribose) polymerase.

Poly(ADP-ribosylation) of histones H1, H5 and non-histone chromosomal high-mobility-group proteins HMG 1, 2, 14 and 17 from chicken erythrocytes by purified calf thymus poly(ADP-ribose) polymerase was studied using acid/urea/Triton gel electrophoresis and autoradiography. With histone H1, besides ADP-ribosylated H1 supporting short chains of polymer, the appearance of H1 'dimer' was observed and this reaction was dependent on NAD concentration and incubation time. In addition, highly modified and/or aggregated species of histone H1 were observed. Histone H5 was slightly ADP-ribosylated at low NAD concentrations. At higher NAD concentrations or after longer incubations the formation of H5 'dimer' and of more modified forms of H5 could be observed. HMG 1 and HMG 2 were found to be ADP-ribosylated, the reaction being dependent on NAD concentration and time. Here again some discrete intermediates appeared. HMG 14 and HMG 17 were only slightly ADP-ribosylated under our experimental conditions. These results indicate that the purified DNA-independent poly(ADP-ribose) polymerase can catalyse the formation of H1 'dimer' as in nuclei and nucleosomes and that H5 and HMG proteins can also be ADP-ribosylated and produce well-defined higher complexes. These modifications of nuclear proteins may provide a means of localized conformational changes of the chromatin structure in vivo.

Animals↗
PubMed1981

Core particle stability critically depends upon a small number of terminal nucleotides.

An apparently homogeneous population of core particles is in fact composed of three subpopulations which behave differently when exposed to a high concentration of ethidium bromide or to 0.6 M NaCl. These subspecies have been identified by the use of several techniques, viz., electron microscopy, sedimentation velocity and circular dichroism. The electrophoretic analysis of their DNA leads to the conclusion that core particle stability critically depends upon a small number of terminal nucleotides.

Animals↗
PubMed1980

Isolation and physical characterization of a stable core particle.

Core particles were prepared from mature chicken erythrocytes chromatin, according to the method of Lutter (J. Mol. Biol. 124, 391, 1978) with one major modification: after the second digestion, zonal centrifugation was used to isolate the core particle, instead of chromatography on Sepharose 6B. By using circular dichroism and electron microscopy, we were able to follow each step of the preparation and to offer an explanation of the discrepancies found in previous preparations and in our own preparations.

Animals↗
PubMed1980

Thermal transition of core particle is not a two-state process.

Thermal transition of core particle which occurs before melting of DNA and can be followed by circular dichroism is not a two-state process; it is the result of two processes which cannot be dissociated in static experiments: unfolding of core particles is immediately followed by their aggregation. It is thus impossible to get thermodynamic parameters of core particle unfolding from its thermal transition monitored by circular dichroism. Thermal denaturation kinetics of core particles gives some information about their stability. Finally core particle structure is more stable in chromatin than in its isolated state.

Journal Article↗
PubMed1980

Distribution of antigenicity in chicken erythrocyte histone H5.

We have compared the inhibitory strengths of eight peptides from chicken histone H5 to that of the parent protein (189 residues) in complement fixation by guinea pig anti-H5 serum complexed with the homologous histone. Two precisely delineated regions (residues 59 to 65 and 94 to 99) and two less-defined regions (between 66 and 93; 100 and 189) are depicted. The sequences of particular consequences are quite conserved in avian histone H5 while the most variable N-terminal portion of 31 residues has no inhibitory effect.

Amino Acid Sequence↗
PubMed1979

Ethidium bromide binding to core particle: comparison with native chromatin.

Ethidium bromide intercalation into DNA of nuclease digested erythrocyte chromatin and core particle, was followed at low ionic strength by fluorescence measurements, equilibrium dialysis using 14C labelled dye, circular dichroism and electron microscopy. High affinity binding sites in the chromatin are no more present in the core particle, i.e. when the linker is removed. In the case of core particle, a cooperative process occurs, accompanied by a partial stripping of the DNA from the core histone. Finally two populations of core particles can be detected by electron microscopy as far as their binding properties are concerned.

Animals↗