G de Murcia

Overproduction of the poly(ADP-ribose) polymerase DNA-binding domain blocks alkylation-induced DNA repair synthesis in mammalian cells.

The zinc‐finger DNA‐binding domain (DBD) of poly (ADP‐ribose) polymerase (PARP, EC 2.4.2.30) specifically recognizes DNA strand breaks induced by various DNA‐damaging agents in eukaryotes. This, in turn, triggers the synthesis of polymers of ADP‐ribose linked to nuclear proteins during DNA repair. The 46 kDa DBD of human PARP, and several derivatives thereof mutated in its first or second zinc‐finger, were overproduced in Escherichia coli, in CV‐1 monkey cells or in human fibroblasts to study their DNA‐binding properties, the trans‐dominant inhibition of resident PARP activity, and the consequences on DNA repair, respectively. A positive correlation was found between the in vitro DNA‐binding capacity of the recombinant DBD polypeptides and their inhibitory effect on PARP activity stimulated by the alkylating agent N‐methyl‐N′‐nitro‐N‐nitrosoguanidine (MNNG). Furthermore, overproduced wild‐type DBD blocked unscheduled DNA synthesis induced in living cells by MNNG treatment, but not that induced by UV irradiation. These results define a critical role for the second zinc‐finger of PARP for DNA single‐stranded break binding and furthermore underscore the importance for PARP to act as a critical regulatory component in the repair of DNA damage induced by alkylating agents.

The human poly(ADP-ribose) polymerase nuclear localization signal is a bipartite element functionally separate from DNA binding and catalytic activity.

Poly(ADP‐ribose) polymerase (PARP, EC 2.4.2.30) is a zinc finger DNA‐binding protein involved in DNA repair processes in eukaryotes. By deletion and extensive site‐directed mutagenesis, its DNA‐binding domain fused to the N‐terminus of beta‐galactosidase was shown to contain a nuclear localization signal (NLS) of the form KRK‐X(11)‐KKKSKK (residues 207–226). In vitro, both the DNA‐binding capacity and the polymerizing activity of PARP are independent of the nuclear location function. Each basic cluster is essential but not sufficient on its own for this function, while both motifs together are. Crucial basic amino acids (K207, R208 and K222) in each of these two motifs are required for nuclear homing. The results presented here support the concept that the human PARP NLS is an autonomous functional element and belongs to the class of bipartite NLSs. We show that the linear distance between the two basic clusters is not crucial. Insertional mutation analysis leading to a partial reversion of the cytoplasmic phenotype displayed by the mutant K222I highlights the crucial positioning of this lysine. The structure‐function relationship of the second cluster of basic residues is discussed.

Electron microscopic visualization of N-acetoxy-N-2-acetylaminofluorene binding sites in ColE1 DNA by means of specific antibodies

ColE1 DNA has been allowed to react in vitro with N-acetoxy-N-2-[14C]acetylaminofluorene in the range of 0-15 N-2-[14C]acetylaminofluorene residues bound per molecule of DNA, at the C8 of guanine residues. Purified rabbit antibodies to both N-2-(guanosine-8-yl)-acetylaminofluorene and native DNA that had reacted with N-acetoxy-N-2-acetylaminofluorene were shown by electron microscopy to recognize specifically the acetylaminofluorene-modified ColE1 DNA. The antibodies bound to DNA were visualized either per se or after reaction with goat anti-rabbit immunoglobulins coupled with ferritin. There was a linear relationship between the average number of antibodies bound per DNA molecule and the number of N-2-(deoxyguanosine-8yl)-acetylaminofluorene residues per DNA molecule. The slope of this straight line was equal to 0.4. Due to the bivalence of the immunoglobulins one would expect a value of 0.5; we actually observed an important fraction of the bound antibodies crosslinking two parts of the same (or of another) DNA molecule.

Use of two-dimensional thin-layer chromatography for the components study of poly(adenosine diphosphate ribose)

Two-dimensional thin-layer chromatography on cellulose plates has been used for separating and quantifying the three adenosine derivatives: AMP, phosphoribosyl AMP (PRAMP), and (PR)2AMP obtained by venom phosphodiesterase digestion of poly(ADP-ribose). In vitro synthesized polymer, up to 300 derivatives in length were studied. Some parameters of the complexity of poly(ADP-ribose) could be deduced from our results: (i) The first branching point appears in fragments of approximately 21 derivatives in length. (ii) The branching points are located at regular distances of approximately 41 derivatives from each other.

Visualization of poly(ADP-ribose) synthetase associated with polynucleosomes by immunoelectron microscopy

Antibodies showing a high specificity for poly(ADP ribose) synthetase have been purified. A fraction binding nonspecifically to histones present in antiserum and non-immune serum has been demonstrated by immunoblotting and separated by histone-Sepharose chromatography. The antibody without the nonspecific binding fraction was analyzed by Western blot with calf thymus protein extract and was found to react only with a band at 116 kDa. There was no reaction with purified topoisomerase I, this weak activity was copurified with poly(ADP-ribose) synthetase preparation. The specific IgG fraction has been used for the visualization of the interaction of poly(ADP-ribose) synthetase with chromatin by indirect gold-labelling. This immunomicroscopic study suggests that the synthetase is located in the inner part of polynucleosomes and would be associated preferentially with the core nucleosome.

Unwinding of supercoiled Col E1-DNA after covalent binding of the ultimate carcinogen N-acetoxy-N-2-acetylaminofluorene and its 7-iodo derivative

The unwinding of superhelical Col E1-DNA was studied by means of gel electrophoresis and electron microscopy after covalent binding of N-acetoxy-N-2-[14C]acetylaminofluorene (N-Aco-[14C]AAF) and its 7-iodo derivative (N-Aco-[14C]AAIF). Studies with both compounds indicated that complete unwinding of the supercoiled DNA required the binding of hydrocarbon residue to about 3% of the bases. Thus the unwinding angle per residue of N-2-acetylaminofluorene (AAF) and its 7-iodo derivative was of 22 degrees +/- 3 and 18 degrees +/- 3 respectively. Our results are in good agreement with those obtained by Drinkwater et al. [9]. Precedent studies from this laboratory have shown that N-Aco-AAF and its 7-iodo derivative induce different local conformation change in native DNA (insertion-denaturation model and outside binding model respectively). The unexpected ability of the 7-iodo derivative to unwind supercoiled DNA is discussed.

Effect of tetramethylammonium ions on conformational changes of DNA in the premelting temperature range

The reversible conformational change of DNAs and polydeoxyribonucleotides occurring before melting was followed by circular dichroism. deltatheta/deltaT, the rate of change of ellipticity theta with temperature, was used mainly as a measure of this premelting phenomenon. If sodium ions were replaced by tetramethylammonium ions deltatheta/deltaT decreased for poly (dA) poly (dT) and poly (dA.dT) poly (dT.dA), but increased for poly (dG.dC) poly (dC.dG). DNAs of different base composition showed no more premelting (deltatheta/deltaT approximately 0) even at low molarities of TMACl provided the Na/TMA ratio was very small. For all cases studied the theta values at 0 degrees C and at a given ionic strength were smaller in NaCl than in TMACl. When studying the series of ammonium ions from NH4+ to (C2H5)4N+, the deltatheta/deltaT values first decreased, going through zero with TMA+ ions, and then increased again. A tentative and qualitative explanation of our results can be given: (a) Hydration of the polymers increases in presence of TMA ions and their average stability decreases; locally, however, (AT) pairs are preferentially stabilized by TMA ions owing to a specific interaction at the level of O2 of thymine. (b) In order to explain the different behaviour of (AT) polymers and DNA, it is assumed that only the B structure is able to accommodate TMA ions in the small groove of the double stranded helix.

A novel form of ataxia oculomotor apraxia characterized by oxidative stress and apoptosis resistance

Several different autosomal recessive genetic disorders characterized by ataxia with oculomotor apraxia (AOA) have been identified with the unifying feature of defective DNA damage recognition and/or repair. We describe here the characterization of a novel form of AOA showing increased sensitivity to agents that cause single-strand breaks (SSBs) in DNA but having no gross defect in the repair of these breaks. Evidence for the presence of residual SSBs in DNA was provided by dramatically increased levels of poly (ADP-ribose)polymerase (PARP-1) auto-poly (ADP-ribosyl)ation, the detection of increased levels of reactive oxygen/nitrogen species (ROS/RNS) and oxidative damage to DNA in the patient cells. There was also evidence for oxidative damage to proteins and lipids. Although these cells were hypersensitive to DNA damaging agents, the mode of death was not by apoptosis. These cells were also resistant to TRAIL-induced death. Consistent with these observations, failure to observe a decrease in mitochondrial membrane potential, reduced cytochrome c release and defective apoptosis-inducing factor translocation to the nucleus was observed. Apoptosis resistance and PARP-1 hyperactivation were overcome by incubating the patients cells with antioxidants. These results provide evidence for a novel form of AOA characterized by sensitivity to DNA damaging agents, oxidative stress, PARP-1 hyperactivation but resistance to apoptosis.

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.

Electron Microscopic Visualization of Chromatin Structure at Sites of DNA Excision Repair Following Ultraviolet Irradiation of Cultured Human Fibroblasts

The DNA excision repair process involves transient changes in chromatin structure at the nucleosomal level (Smerdon and Lieberman, 1978; Smerdon, et al., 1978; Tlsty and Lieberman, 1978; Smerdon, et al., 1979; Oleson, et al., 1979; Smerdon and Lieberman, 1980; Bodell and Cleaver, 1981; Bodell, et al., 1982; Zolan, et al., 1982). Although there is no direct evidence for an early disruption step, a late step, termed nucleosome rearrangement, has been identified which results in the restoration of the native nucleosomal structure at the sites of excision repair. This step has the following characteristics: repair incorporated nucleotides are initially very sensitive to digestion by staphylococcal nuclease and DNase I but eventually acquire the same sensitivity as nucleotides in bulk chromatin (Smerdon and Lieberman, 1978; Smerdon, et al., 1978; Tlsty and Lieberman, 1978; Smerdon, et al., 1979; Oleson, et al.. 1979; Smerdon and Lieberman, 1980; Bodell and Cleaver, 1981; Bodell, et al., 1982; Zolan, et al., 1982); and repair incorporated nucleotides are initially underrepresented in nucleosomal core-length DNA fragments produced by staphylococcal nuclease digestion but eventually acquire a random distribution (Smerdon and Lieberman, 1978; Smerdon, et al., 1978; Tlsty and Lieberman, 1978; Smerdon, et al., 1979; Oleson, et al., 1979; Smerdon and Lieberman, 1980; Bodell and Cleaver, 1982).