Alice Mazen

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.

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.

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.

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.

Chicken erythrocyte histone H5; I amino terminal sequence (70 residues)

In addition to the five histones common to most vertebrate species, a specific histone (histone H5)* occurs in the nucleated erythrocyte of bird, amphibian, reptile and fish. This histone has been isolated from chicken erythrocytes and described by Neelin et al. [l] and Hnilica [2]. Histone H5 is thought to be synthesized in place pf a certain amount of histone Hr in mature chicken erythrocyte, since the two histones together equal the usual amount of histone Hr present in the other tissues [3]. Although related to each other by their solubility in 5% perchloric acid or trichloracetic acid and by their molecular size, the histones Hr and Hs differ markedly by their amino-acid composition and particularly in their arginine content [4,5]. The amino acid sequence of the N-terminal fragment obtained by cyanogen bromide cleavage of the

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.

Comparison of the DNA repeat length in H1- and H5-containing chromatin (Mature hen erythrocytes, immature chick erythroid cells and hen liver)

The variability of the DNA repeat length in chromatin of eukaryotes is now well documented [ 1-11 ]. The size of the repeating unit ranges from 154 base pairs in Aspergillus nidulans [2] to 241 base pairs in the chromatin of sea urchin sperm [7]. As yet the origin of this phenomenon has not been explained but a few possible causes of the observed variability have been excluded [7-9]; indeed it has been shown that the rate of cell division, the stage in the cell cycle, the genomic activity, the phosphorylation of H1 and the acetylation of histone Ha and H4 could not be correlated to the variation of the repeat length of chromatin. Noll [1 ] and Morris [2] have suggested that there may be a relationship between the structure of historic H1 (as expressed by the content of basic amino acids) and the length of the DNA repeat in chromatin. As a test of this hypothesis Morris [3] has compared the structure of hen liver and hen erythrocyte chromatin in which a large fraction of the histone H1 is replaced by the lysine rich histone Hs; the finding that the repeat length of hen erythrocyte chromatin was longer than that of hen liver chromatin has suggested that there may be a correlation between the increased length of the repeat of hen erythrocyte chromatin and the presence of Hs. In the present work we have compared the DNA repeat length from mature erythroid cells of hen, immature erythroid cells of three-day old chicks [I 2] and hen liver chromatin. We show that the immature chick erythroid cell chromatin has the same repeat length as the hen liver chromatin, both being smaller than that of adult hen erythrocyte chromatin. Since the erythroid cells of chick contain the histone Hs whereas the hen liver cells do not, our results suggest that there is no direct relationship between the presence of Hs and the increased length of the repeating unit in the hen erythrocytes.

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.

The structure of Sipunculus nudus erythrocyte chromatin

Abstract The structure of the Sipunculus erythrocyte chromatin has been characterized by electron microscopy and nuclease digestion (staphylococcal nuclease and pancreatic nuclease). Contrary to previous results [1], we were able to isolate and characterize a histone H2B in sipunculid nuclei. Though the histones H2A and H2B were markedly different from their vertebrate homologues, the subunit structure of the chromatin is the same. But the length of the repeat unit of DNA in the chromatin, is 177 ± 5 bp for the sipunculid erythrocyte nuclei, close to that reported for the chromatin of some lower eukaryotes.

Non-random binding of N-acetoxy-N-2-acetylaminofluorene 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.