Claude Niedergang

XRCC1 Is Specifically Associated with Poly(ADP-Ribose) Polymerase and Negatively Regulates Its Activity following DNA Damage

ABSTRACT 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 theSaccharomyces 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 β, 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.

Structure and function of poly(ADP-ribose) polymerase

Poly(ADP-ribose) polymerase (PARP) participates in the intricate network of systems developed by the eukaryotic cell to cope with the numerous environmental and endogenous genetoxic agents. Cloning of the PARP gene has allowed the development of genetic and molecular approaches to elucidate the structure and the function of this abundant and highly conserved enzyme. This article summarizes our present knowledge in this field.

Poly(adenosine diphosphate ribose)

Publisher Summary This chapter discusses the higher-order structure of poly(adenosine diphosphate ribose) [poly(ADPR)], poly(ADPR) polymerase purification and its properties, and molecular mechanisms of histone- and elongation-factor (EF-2)-ADP-ribosylation. The precise biological function of the poly(ADP-ribosylation) reaction has not yet been unequivocally established. However, the results are consistent with the occurrence of rapid transient modifications of proteins in localized regions of chromatin. These alterations in chromatin conformation appear to be induced by DNA strand breakage leading to the DNA repair process or to the differentiation process. Thus, after analytical studies in vitro —such as enzyme purification and properties determination and enzyme activity measurements in isolated nuclei—the use of new sensitive analytical techniques in vivo should allow a better understanding of the biological functions of mono and poly(ADPR). The chapter also discusses the process of DNA-repair as one of the biological functions of poly(ADPR) polymerase and presents new techniques for poly(ADPR) studies including immunological analysis.

A dual approach in the study of poly (ADP-ribose) polymerase: In vitro random mutagenesis and generation of deficient mice

A dual approach to the study of poly (ADP-ribose)polymerase (PARP) in terms of its structure and function has been developed in our laboratory. Random mutagenesis of the DNA binding domain and catalytic domain of the human PARP, has allowed us to identify residues that are crucial for its enzymatic activity.

Poly(ADPribose) levels as a function of chick limb mesenchymal cell development as studied in vitro and in vivo

Abstract NAD is converted into a chromatin-bound polymer, poly(ADPribose), with the excision of nicotinamide. In intact cells, the incorporation of labeled adenine, through NAD, into poly(ADPribose) has been correlated with the commitment and/or initial phenotypic expression of chick limb mesenchymal cells. Using an assay for chemical quantities of poly(ADPribose), we report here measurements of poly(ADPribose) during limb development in situ and during limb mesenchymal cell commitment and expressional events in cell culture. Substantial changes in the levels of poly(ADPribose) are observed during early phases of limb cell development either in situ (embryonic stages 22 to 26) or in culture (Days 1 to 4); during this time, we observed a threefold decrease in poly(ADPribose) per unit DNA (21 to 7 nmoles/mg DNA), as compared to relatively minor changes of 10 to 20% during later expressional events especially related to muscle development. These observations establish a correlation between cellular poly(ADPribose) levels and the early phases of chick limb mesenchymal cell differentiation and development.

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.

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.

Poly ADP-ribose polymerase: Self-ADP-ribosylation, the stimulation by DNA, and the effects on nucleosome formation and stability

Abstract A partially purified preparation of the enzyme poly ADP-ribose polymerase which controls the transfer of ADP-ribose from NAD has been investigated. Data presented here indicate that the enzyme ADP-ribosylates itself. The enzyme preparation can be stimulated by DNA and this stimulation is exclusively associated with an auxiliary protein which copurifies with the enzyme and which we refer to as endogenous acceptor protein. Exogenously added proteins such as histones H1, H2A, and H3, cholera toxin, and Escherichia coli DNA-dependent RNA polymerase can also act as acceptor proteins in addition to the DNA-associated labeling of the endogenous acceptor. We speculate that the self-ADP-ribosylation of enzyme and that of the endogenous acceptor may play a role in control of the extremely rapid turnover of cellular NAD. Additionally, we have used this enzyme to ADP-ribosylate histones and to determine the effect of such modification on in vitro nucleosome formation and stability. The enzyme mediated ADP-ribosylation of free histones prior to incorporation into nucleosomes affects both nucleosome formation and stability while such ADP-ribosylation of histones already incorporated into nucleosomes does not affect their stability. These observations suggest that the ADP-ribosylation of histones prior to their involvement in nucleosomes might be the site of the physiologically important ADP-ribose transfer.

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.

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.