XRCC1 is specifically associated with poly(ADP-ribose) polymerase and negatively regulates its activity following DNA damage.

Poly(ADP-ribose) polymerase (PARP; EC 2.4.2.30) is a zinc-finger DNA-binding protein that detects and signals DNA strand breaks generated directly or indirectly by genotoxic agents. In response to these breaks, the immediate poly(ADP-ribosyl)ation of nuclear proteins involved in chromatin architecture and DNA metabolism converts DNA damage into intracellular signals that can activate DNA repair programs or cell death options. To have greater insight into the physiological function of this enzyme, we have used the two-hybrid system to find genes encoding proteins putatively interacting with PARP. We have identified a physical association between PARP and the base excision repair (BER) protein XRCC1 (X-ray repair cross-complementing 1) in the Saccharomyces cerevisiae system, which was further confirmed to exist in mammalian cells. XRCC1 interacts with PARP by its central region (amino acids 301 to 402), which contains a BRCT (BRCA1 C terminus) module, a widespread motif in DNA repair and DNA damage-responsive cell cycle checkpoint proteins. Overexpression of XRCC1 in Cos-7 or HeLa cells dramatically decreases PARP activity in vivo, reinforcing the potential protective function of PARP at DNA breaks. Given that XRCC1 is also associated with DNA ligase III via a second BRCT module and with DNA polymerase beta, our results provide strong evidence that PARP is a member of a BER multiprotein complex involved in the detection of DNA interruptions and possibly in the recruitment of XRCC1 and its partners for efficient processing of these breaks in a coordinated manner. The modular organizations of these interactors, associated with small conserved domains, may contribute to increasing the efficiency of the overall pathway.

Poly(ADPR)polymerase expression and activity during proliferation and differentiation of rat astrocyte and neuronal cultures.

Poly(ADPR)polymerase (poly(ADPR)P) mRNA and enzymatic activity levels were investigated in primary cultures of rat astrocytes and neurons in the absence or presence of basic fibroblast growth factor (bFGF) and nerve growth factor (NGF), respectively. In cultured rat astrocytes, a biphasic increase in poly(ADPR)P mRNA, associated with enhanced nuclear poly(ADPR)P enzymatic activity, were observed. The first rise in poly(ADPR)P mRNA and enzymatic activity is at the beginning of cell proliferation and the second with the occurrence of cell differentiation. In the presence of bFGF (5 ng/ml) the mRNA peaks and the differentiation-associated poly(ADPR)P enzymatic activity undergoes a 2-fold increase. In neuronal cultures an initial high level of poly(ADPR)P mRNA is followed by a decrease while differentiation is progressively achieved. A limited increase of poly(ADPR)P activity is observed during this phase. In the presence of NGF (50 ng/ml), similar poly(ADPR)P mRNA expression and enzymatic activity patterns are observed. The results suggest that poly(ADPR)P is involved at the onset of nerve-cell proliferation and differentiation.

Purification of an alkaline ribonuclease from soluble fraction of beef brain.

A ribonuclease (ribonucleate 3-pyrimidine-oligonucleotidohydrolase, EC 3.1.4.22) was purified 8300-fold from soluble fraction of beef brain and its properties were investigated. The enzyme is an endonuclease capable of hydrolyzing tRNA, rRNA, poly(C), but shows no activity towards poly(U), poly(A), and poly(G). The preparation is free of deoxyribonuclease, non-specific phosphodiesterase and phosphomonoesterase activity. The enzyme has a pH optimum of 7.6, is not heat stable, has a molecular weight of 25 000, and has a K-m of 134 mu rRNA and K-m of 1600 mug poly(C) per ml.

Crystallization and X-ray crystallographic analysis of recombinant chicken poly(ADP-ribose) polymerase catalytic domain produced in Sf9 insect cells.

Poly (ADP-ribose) polymerase (PARP) participates in the immediate response in mammalian cells exposed to DNA-damaging agents. Recombinant baculovirus harboring the cDNA of the chicken PARP catalytic domain (40 kDa) have been used to infect Spodoptera frugiperda (Sf9) insect cells. The recombinant polypeptide (30 mg per 1 x 10(9) cells) was purified to homogeneity by 3-aminobenzamide affinity chromatography. The enzymatic properties of the recombinant domain were similar to those of the native fragment. Crystals of the purified recombinant catalytic domain were grown by vapor diffusion. The crystals belong to space group P2(1)2(1)2(1) with unit cell dimensions of a = 59.2 A, b = 65.0 A, c = 96.9 A. They are suitable for X-ray analysis and diffract to 2.0 A.

Chicken poly(ADP-ribose) synthetase: complete deduced amino acid sequence and comparison with mammalian enzyme sequences.

The complete nucleotide (nt) sequence of the cDNA encoding the chicken poly(ADP-ribose) synthetase has been determined. Positive clones overlapping the 5' region or the 3' region of the cDNA have been isolated from a lambda gt 10 hen oviduct cDNA library using two human cDNA probes. The missing middle portion has been obtained by the polymerase chain reaction procedure. A single 3033-nt open reading frame from start codon to stop codon encodes a sequence of 1011 amino acid residues. The alignment of this sequence with those from human and mouse reveals overall identities of 79% and 77%, respectively. However, an identity of about 82% is obtained in the DNA-binding domain within the two zinc fingers, and an even higher similarity (85-87%) is observed in the NAD-binding domain. The isolated clones consistently hybridize on chicken Northern blots to an mRNA species of about 4 kb, whereas they do not cross-hybridize with RNA blots of Drosophila melanogaster. Thus, it appears that, even if the functional properties of the enzyme are maintained, the cDNA identity will be much decreased in nonvertebrate organisms.

Poly(ADP-ribose) polymerase interacts with a novel human ubiquitin conjugating enzyme: hUbc9.

Poly(ADP-ribose) polymerase (PARP) has been suggested to play a regulatory role in vivo, in DNA replication and/or DNA repair based mainly on its capacity to bind to DNA strand breaks. This interaction is modulated through auto poly(ADP-ribosylation). However, the biological function of PARP may also involve interactions with proteins such as topoisomerase I or DNA polymerase alpha, which may or may not be themselves ADP-ribosylated. Using the yeast two-hybrid method search for other proteins interacting with PARP, we have isolated a full-length cDNA clone coding for a protein of 158 amino acid residues. This amino acid sequence is 66 and 56% identical to yeast ubiquitin-conjugating enzymes Hus5 and Ubc9 of Schizosaccharomyces pombe and Saccharomyces cerevisiae, respectively. Moreover, we have demonstrated that the expressed protein complements a S. cerevisiae yeast strain deficient for Ubc9. The protein encoded by the isolated cDNA is thus a new human counterpart of the ubiquitin-conjugating enzyme family and has been called hUbc9. The hubc9 gene locus has been assigned to the chromosomal location 16p13.2-p13.3. By means of two-hybrid analysis it was discovered that hUbc9 interacts with the automodification domain of PARP. This interaction was further confirmed using GST (glutathione-S-transferase) tagged fusion proteins: (i) in vivo, by transfecting cos7 cells with hUbc9 cloned in an eukaryotic expression vector, and (ii) in vitro, by mixing purified PARP with hUbc9 purified and expressed in bacteria. The possible significance and function of this interaction is discussed while taking into account the possible intracellular role of hUbc9.

Adenosine diphosphate ribosylation of histone H1 by purified calf thymus polyadenosine diphosphate ribose polymerase.

The mechanism of poly ADPR synthesis and the transfer of poly ADPR to histone H1 molecule by electrophoretically homogenous calf thymus poly ADPR polymerase containing DNA was examined. 1) An acid insoluble radioactive complex (I) was obtained after incubation of purified enzyme with [3H] NAD. The stability of (I) was examined by SDS-polyacrylamide gel electrophoresis. The complex (I) was stable against acid, SDS, urea, DNase and RNase, but labile against pronase, trypsin, alkali and snake venom phosphodiesterase treatment. The molecular weight of (I) was about 130 000 daltons estimated by SDS-gel electrophoresis. The radioactive products of successive alkali, venom phosphodiesterase and Pronase hydrolysis of (I) were PR-AMP and AMP. The mean chain length of poly ADPR of (I) was 20--30. These results suggest that the complex (I) is poly ADP-ribosylated poly ADPR polymerase. 2) Besides (I), a second radioactive peak (II) was observed when acid insoluble products obtained from an incubation mixture containing purified poly ADPR polymerase, [3H] NAD and purified histone H1 were analyzed on SDS-polyacrylamide gel electrophoresis. The molecular weight of (II) was estimated to be about 23 000 daltons. The complex (II) is eluted like histone H1 on CM-cellulose columns and hydrolyzed by alkali, trypsin and snake venom phosphodiesterase but not by DNase, or RNase. The comples (II) was extracted selectively by 5 per cent perchloric acid or 5 per cent trichloroacetic acid from mixture of (I) and (II). The mean chain length of poly ADPR of complex (II) and 5--20; these results suggest that the complex (II) is poly ADP-ribosylated histone H1. 3) Results 1) and 2) indicate that purified DNA containing, thus DNA independent, poly ADPR polymerase catalyzes two different reactions, the ADPR transfer onto the enzyme itself and onto histone H1 and the elongation of ADPR chains. Dimeric forms of ADP-ribosylated histone H1 was not observed. Free poly ADPR was observed only when very small quantities of enzyme were used for incubation.

Adenosine diphosphate ribosyltransferase and protein acceptors associated with cytoplasmic free messenger ribonucleoprotein particles.

ADP-ribosyltransferase activity has been characterized in free messenger ribonucleoprotein particles (mRNP) from mouse plasmacytoma cells. This enzymatic activity appears to be associated with the free mRNP and not due to nuclear contamination. The enzyme activity is not stimulated by added DNA or histone H1 and represents 34 per cent of the total cellular ADP-ribosyltransferase activity while the DNA contamination in free mRNP is less than 4 per cent of the total cellular DNA. Moreover, the ADP-ribosyltransferase specific activity per mg of DNA is about 75-fold higher in free mRNP than in the nuclei. During CsCl gradient centrifugation of the cytoplasmic fraction, the ADP-ribosylated material separates out at a buoyant density similar to that of free mRNP. This ADP-ribosyltransferase activity is inhibited by thymidine, nicotinamide and 3-aminobenzamide, while it is highly stimulated by exogenous pancreatic RNase. The in vitro synthesized acid insoluble material is rendered partly soluble by treatment by a proteolytic enzyme or by snake venom phosphodiesterase resulting in phosphoribosyl-AMP formation: the pancreatic RNase does not solubilize this material. Several ADP-ribosylated proteins are detected by lithium dodecylsulfate gel electrophoresis. Such an ADP-ribosyltransferase activity has also been detected in free mRNP from rat liver. It is suggested that this ADP-ribosylation of specific free mRNP proteins may be associated with free mRNP structure and/or with some chemical covalent type of modification rendering mRNA available for translation.

Poly(ADP-ribose) polymerase: structure-function relationship.

Dissection of the human poly(ADP-ribose) polymerase (PARP) molecule in terms of its structure-function relationship has proved to be an essential step towards understanding the biological role of poly(ADP-ribosylation) as a cellular response to DNA damage in eukaryotes. Current approaches aimed at elucidating the implication of this multifunctional enzyme in the maintenance of the genomic integrity will be presented.

Involvement of poly(ADP-ribose) polymerase in base excision repair.

Poly(ADP-ribose) polymerase (PARP) is a zinc-finger DNA binding protein that detects and signals DNA strand breaks generated directly or indirectly by genotoxic agents. In response to these lesions, the immediate poly(ADP-ribosylation) of nuclear proteins converts DNA interruptions into intracellular signals that activate DNA repair or cell death programs. To elucidate the biological function of PARP in vivo, the mouse PARP gene was inactivated by homologous recombination to generate mice lacking a functional PARP gene. PARP knockout mice and the derived mouse embryonic fibroblasts (MEFs) were acutely sensitive to monofunctional alkylating agents and gamma-irradiation demonstrating that PARP is involved in recovery from DNA damage that triggers the base excision repair (BER) process. To address the issue of the role of PARP in BER, the ability of PARP-deficient mammalian cell extracts to repair a single abasic site present on a circular duplex plasmid molecule was tested in a standard in vitro repair assay. The results clearly demonstrate, for the first time, the involvement of PARP in the DNA synthesis step of the base excision repair process.

Characterization of the poly(ADP-ribose) polymerase associated with free cytoplasmic mRNA-protein particles.

Poly(ADP-ribose) polymerase associated with free cytoplasmic messenger ribonucleoprotein particles (mRNP) has been characterized in mouse plasmacytoma. This cytoplasmic enzyme undergoes auto-ADP-ribosylation and has a similar molecular weight and common antigenic sites with the chromatin bound poly(ADP-ribose) polymerase in spite of its DNA independency. The free mRNP poly(ADP-ribose) polymerase is released from the particle only by high saline concentrations (0.7 M KCl) and the dissociated enzyme expresses a higher activity. The treatment of free mRNP by RNase A stimulates the poly(ADP-ribose) polymerase activity. Partial destruction of mRNP by high saline concentration or mRNA digestion unmasks new protein sites for ADP-ribosylation. In view of the changes that occur in the free mRNP structure to permit mRNA translation, a possible role of poly(ADP-ribosylation) as an important post-synthetic modification of some of the mRNP proteins is discussed.

Properties of purified calf thymus poly(adenosine diphosphate ribose) polymerase. Comparison of the DNA-independent and the DNA-dependent enzyme.

The physicochemical properties of the purified calf thymus poly(ADP-ribose) polymerase were investigated. The enzyme purified to homogeneity was shown to contain about 10% DNA on a weight basis and its activity to be DNA independent. After removing this fragment of DNA, called the sDNA fraction, the enzyme becomes DNA dependent. The activity of this enzyme preparation was entirely dependent on, and completely restored by, added calf thymus DNA or sDNA. However, the calf thymus DNA concentration needed was a hundred times higher than that of sDNA. The properties of the two enzyme preparations, DNA independent and DNA dependent, were essentially the same. They both reacted against the specific antibody obtained with the DNA-independent poly(ADP-ribose) polymerase. The pH optimum was around 8; the activity was stimulated by Mg2+, Mn2+ and Ca2+, and inhibited by high ionic strength, p-chloromercuribenzoate, ADP-ribose, AMP and polylysine. Nicotinamide, thymidine and NADP were shown to be competitive inhibitors. The enzymatic activity was stimulated by histone H1 when the ratio of DNA to histone H1 was 2. Histones H2A, H2B, H3 and H4 had little effect on the DNA-independent enzyme activity, but were strongly inhibitory for the DNA-dependent enzyme. This inhibitory effect could be reversed by allowing the DNA-dependent enzyme to react with the sDNA fraction before adding histone subfractions. The apparent Km for NAD of the DNA-dependent poly(ADP-ribose) polymerase was shown to vary with the DNA concentration. It was minimum when the amount of sDNA was 10% of that of the enzyme. The ratio of the apparent Km for sDNA to the enzyme concentration was constant at any enzyme concentration. The minimum estimation of the number of base pairs of sDNA required for maximal activation of one enzyme molecule was 16. For calf thymus DNA, this estimation was of 640. These results suggest that the activation of the enzyme needs the formation of some complex between the protein and a specific part of the DNA. This complex was preserved in the DNA-independent enzyme preparation.

[Purification of calf thymus adenosine diphosphate ribose polymerase]. / Purification de l'adénosine diphosphate ribose polymérase de thymus de Veau.

Calf thymus poly ADPR polymerase has been purified to electrophoretic homogenity. The enzyme has a molecular weight of 120,000 +/- 10,000 dalton. The substrate affinity is very high (apparent Km 82.5 micrometer). The presence of exogenous DNA does not appear to be a requisite for enzymatic activity of the purified enzyme.

DNA topoisomerase I from calf thymus is inhibited in vitro by poly(ADP-ribosylation).

A slight DNA topoisomerase I activity was detected in highly purified poly(ADP-Rib)polymerase prepared from calf thymus. This copurified activity was found to be suppressed under conditions where the poly(ADP-ribosylation) reaction occurs in the presence of NAD. Purified topoisomerase I from calf thymus was shown to be ADP-ribosylated by poly(ADP-Rib) polymerase purified from the same tissue. Poly(ADP-ribosylation) of topoisomerase I produces an inhibition of the enzymatic activity in parallel to the extent of ADP-ribosylation. The fact that a slight poly(ADP-Rib) polymerase activity was also found to copurify with a topoisomerase I preparation and that topoisomerase I activity can be modified by ADP-ribosylation, may suggest a spatial and functional correlation of these two enzymes in chromatin.

Sequential ADP-ribosylation pattern of nucleosomal histones. ADP-ribosylation of nucleosomal histones.

The pattern of nucleosomal histones poly(ADP-ribosyl)ation is changed under conditions which affect the poly(ADP-ribosyl)ation state of the enzyme. At low NAD concentrations the enzyme can poly(ADP-ribosyl)ate histones H1 and H1, H2A, A2A, and H2B. However at NAD concentrations above 10 microM the enzyme preferentially poly(ADP-ribosyl)ates histone H1 to a hyper ADP-ribosylated form. Furthermore we have observed hyper ADP-ribosylation of histone H2B at NAD concentrations of 10 microM suggesting that histone H2B can undergo the same type of ADP-ribosylation pattern as histone H1. Also at higher NAD concentrations an elongation of the polymer attached to the enzyme and other nuclear proteins takes place.