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Query: UNIPROT:P06889 (Mol)
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Recent evidence obtained with transgenic knockout mice suggests that the enzyme poly(ADP-ribose)polymerase (PARP) does not play a direct role in DNA break processing. Nevertheless, inactivation of the catalytic or the DNA nick-binding functions of PARP affects cellular responses to genotoxins at the level of cell survival, sister chromatid exchanges and apoptosis. In the present report, we conceptualize the idea that PARP is part of a DNA break signal mechanism. In vitro screening studies revealed the existence of a protein family containing a polymer-binding motif of about 22 amino acids. This motif is present in p53 protein as well as in MARCKS, a protein involved in the regulation of the actin cytoskeleton. Biochemical analyses showed that these sequences are directly targeted by PARP-associated polymers in vitro, and this alters several molecular functions of p53- and MARCKS protein. PARP-deficient knockout mice from transgenic mice were found to exhibit several phenotypic features compatible with altered DNA damage signaling, such as downregulation and lack of responsiveness of p53 protein to genotoxins, and morphological changes compatible with MARCKS-related cytoskeletal dysfunction. The knockout phenotype could be rescued by stable expression of the PARP gene. We propose that PARP-associated polymers may recruit signal proteins to sites of DNA breakage and reprogram their functions.
Mol Cell Biochem 1999 Mar
PMID:Poly ADP-ribosylation: a DNA break signal mechanism. 1033 31

We have developed a novel enzyme assay that allows the simultaneous determination of noncovalent interactions of poly(ADP-ribose) with nuclear proteins as well as poly(ADP-ribose) glycohydrolase (PARG) activity by high resolution polyacrylamide gel electrophoresis. ADP-ribose chains between 2 and 70 residues in size were enzymatically synthesized with pure poly(ADP-ribose) polymerase (PARP) and were purified by affinity chromatography on a boronate resin following alkaline release from protein. This preparation of polymers of ADP-ribose was used as the enzyme substrate for purified PARG. We also obtained the nuclear matrix fraction from rat liver nuclei and measured the enzyme activity of purified PARG in the presence or absence of either histone proteins or nuclear matrix proteins. Both resulted in a marked inhibition of PARG activity as determined by the decrease in the formation of monomeric ADP-ribose. The inhibition of PARG was presumably due to the non-covalent interactions of these proteins with free ADP-ribose polymers. Thus, the presence of histone and nuclear matrix proteins should be taken into consideration when measuring PARG activity.
Mol Cell Biochem 1999 Mar
PMID:Measurement of poly(ADP-ribose) glycohydrolase activity by high resolution polyacrylamide gel electrophoresis: specific inhibition by histones and nuclear matrix proteins. 1033 32

Here, we describe the latest developments on the mechanistic characterization of poly(ADP-ribose) polymerase (PARP) [EC 2.4.2.30], a DNA-dependent enzyme that catalyzes the synthesis of protein-bound ADP-ribose polymers in eucaryotic chromatin. A detailed kinetic analysis of the automodification reaction of PARP in the presence of nicked dsDNA indicates that protein-poly(ADP-ribosyl)ation probably occurs via a sequential mechanism since enzyme-bound ADP-ribose chains are not reaction intermediates. The multiple enzymatic activities catalyzed by PARP (initiation, elongation, branching and self-modification) are the subject of a very complex regulatory mechanism that may involve allosterism. For instance, while the NAD+ concentration determines the average ADP-ribose polymer size (polymerization reaction), the frequency of DNA strand breaks determines the total number of ADP-ribose chains synthesized (initiation reaction). A general discussion of some of the mechanisms that regulate these multiple catalytic activities of PARP is presented below.
Mol Cell Biochem 1999 Mar
PMID:Regulatory mechanisms of poly(ADP-ribose) polymerase. 1033 33

We have studied the clonogenic survival response to X-rays and MNNG of V79 Chinese hamster cells and two derivative cell lines, ADPRT54 and ADPRT351, deficient in poly(ADP-ribose) polymerase (PARP) activity. Under conditions of exponential growth, both PARP-deficient cell lines are hypersensitive to X-rays and MNNG compared to their parental V79 cells. In contrast, under growth-arrested, confluent conditions, V79 and PARP-deficient cells become similarly sensitive to X-rays and MNNG suggesting that PARP may be involved in the repair of X-ray or MNNG-induced DNA damage in logarithmically growing cells but not in growth-arrested confluent cells. This suggestion, however, creates a dilemma as to how PARP can be involved in DNA repair in only selected growth phases while it is functionally active in all growth phases. To explain these paradoxical results and resolve this dilemma we propose a hypothesis based on the consistent observation that inhibition of PARP results in a significant increase in sister chromatid exchange (SCEs). Thus, we propose that PARP is a guardian of the genome that protects against DNA recombination. We have extended this theme to provide an explanation for our results and the studies done by many others.
Mol Cell Biochem 1999 Mar
PMID:Poly(ADP-ribose) polymerase: a guardian of the genome that facilitates DNA repair by protecting against DNA recombination. 1033 34

In most eukaryotic cells, the catalytic activation of poly(ADP-ribose) polymerase (PARP) represents one of the earliest cellular responses to the infliction of DNA damage. To study the biological function(s) of poly(ADP-ribosyl)ation, we have established stable transfectants (COM3 cells) of the SV40-transformed Chinese hamster cell line C060 which conditionally overexpress the PARP DNA-binding domain upon addition of dexamethasone. We could demonstrate that DNA-binding domain overexpression, which leads to trans-dominant inhibition of poly(ADP-ribosyl)ation, potentiates the cytotoxicity of alkylation treatment and of gamma-radiation. Likewise, carcinogen-induced gene amplification, viewed as a manifestation of genomic instability, was potentiated by the overexpression of the PARP DNA-binding domain. Recently, we studied the effect of trans-dominant PARP inhibition on mutagenesis by employing a shuttle-vector assay in which mutagen-exposed plasmid pYZ289 is electroporated into COM3 cells. We could show that dexamethasone-induced overexpression of the PARP DNA-binding domain in COM3 cells potentiates the mutagenicity of the alkylating agent N-methyl-N-nitrosourea, while no effect of dexamethasone treatment on mutation frequency was recorded in control cells lacking the PARP DNA-binding domain transgene. Taken together, our results further substantiate the role of poly(ADP-ribosyl)ation in the maintenance of genomic integrity and stability under conditions of genotoxic stress.
Mol Cell Biochem 1999 Mar
PMID:Trans-dominant inhibition of poly(ADP-ribosyl)ation potentiates alkylation-induced shuttle-vector mutagenesis in Chinese hamster cells. 1033 35

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. In parallel PARP knock-out mice were generated by inactivation of both alleles by gene targeting. We showed that: (i) they are exquisitely sensitive to gamma-irradiation, (ii) they died rapidly from acute radiation toxicity to the small intestine, (iii) they displayed a high genomic instability to gamma-irradiation and MNU injection and, (iv) bone marrow cells rapidly underwent apoptosis following MNU treatment, demonstrating that PARP is a survival factor playing an essential and positive role during DNA damage recovery and survival.
Mol Cell Biochem 1999 Mar
PMID:A dual approach in the study of poly (ADP-ribose) polymerase: in vitro random mutagenesis and generation of deficient mice. 1033 38

We have recently described the isolation and characterization of bovine cDNA encoding poly(ADP-ribose) glycohydrolase (PARG). We describe here the preparation and characterization of antibodies to PARG. These antibodies have been used to demonstrate the presence of multiple forms of PARG in tissue and cell extracts from bovine, rat, mouse, and insects. Our results indicate that multiple forms of PARG previously reported could result from a single gene. Analysis of PARG in cells in which poly(ADP-ribose) polymerase (PARP) has been genetically inactivated indicates that the cellular content of PARG is regulated independently of PARP.
Mol Cell Biochem 1999 Mar
PMID:Molecular heterogeneity and regulation of poly(ADP-ribose) glycohydrolase. 1033 41

Poly(ADP-ribose) polymerase (PARP) is conserved in eukaryotes. To analyze the function of PARP, we isolated and characterized the gene for PARP in Drosophila melanogaster. The PARP gene consisted of six translatable exons and spanned more than 50 kb. The DNA binding domain is encoded by exons 1-4. Although the consensus cleavage site of CED-3 like protease during apoptosis is conserved from human to Xenopus laevis PARPs, it is neither conserved in the corresponding region of Drosophila nor Sarcophaga peregrina. There are two cDNAs species in Drosophila. One cDNA could encode the full length PARP protein (PARP I), while the other is a truncated cDNA which could encode a partial-length PARP protein (PARP II), which lacks the automodification domain and is possibly produced by alternative splicing. The expression of these two forms of PARP in E. coli demonstrated that while PARP II has the catalytic NAD-binding domain and DNA-binding domain it is enzymatically inactive. On the other hand PARP I is active. A deletion mutant of PARP gene could grow to the end of embryogenesis but did not grow to the adult fly. These results suggest that the PARP gene plays an important function during the development of Drosophila.
Mol Cell Biochem 1999 Mar
PMID:Functional analysis of poly(ADP-ribose) polymerase in Drosophila melanogaster. 1033 45

The concerted action of poly(ADP-ribose) polymerase (PARP) which synthesizes the poly(ADP-ribose) (pADPr) in response to DNA strand breaks and the catabolic enzyme poly(ADP-ribose) glycohydrolase (PARG) determine the level of polymer and the rate of its turnover. In the present study, we have shown that the quail myoblast cells have high levels of basal polymer as compared to the murine C3H10T1/2 fibroblasts. We have conducted this study to investigate how such differences influence polymer synthesis and its catabolism in the cells in response to DNA damage by alkylating agent. In quail myoblast cells, the presence of high MNNG concentration such as 200 microM for 30 min induced a marginal decrease of 15% in the NAD content. For C3H10T1/2 cell line, 64 microM MNNG provoked a depletion of NAD content by approximately 50%. The induction of the polymer synthesis in response to MNNG treatment was 6-fold higher in C3H10T1/2 cells than in quail myoblast cells notwithstanding the fact that 3-fold higher MNNG concentration was used for quail cells. The polymer synthesis thus induced in quail myoblast cells had a 4-5 fold longer half life than those induced in C3H10T1/2 cells. To account for the slow turnover of the polymer in the quail myoblast cells, we compared the activities of the polymer catabolizing enzyme (PARG) in the two cell types. The quail myoblast cells had about 25% less activity of PARG than the murine cells. This difference in activity is not sufficient to explain the large difference of the rate of catabolism between the two cell types implicating other cellular mechanisms in the regulation of pADPr turnover.
Mol Cell Biochem 1999 Mar
PMID:Poly(ADP-ribose) turnover in quail myoblast cells: relation between the polymer level and its catabolism by glycohydrolase. 1033 49

To elucidate the biological functions of poly(ADP-ribose) polymerase (PARP, [EC 2.4.2.30]) in DNA damage responses, genetic and biochemical approaches were undertaken. By disrupting exon 1 of the mouse PARP gene by a homologous recombination, PARP-deficient mouse embryonic stem (ES) cell lines and mice could be produced without demonstrating lethality. PARP-/- ES cells showed complete loss of PARP activity and increased sensitivity to gamma-irradiation and an alkylating agents, indicating a physiological role for PARP in the response to DNA damage. p53, a key molecule in cellular DNA damage response, was found to stimulate PARP activity and became poly(ADP-ribosyl)ated in the presence of damaged DNA. However, PARP-/- ES cells showed p21 and Mdm-2 mRNA induction following gamma-irradiation, indicating that PARP activity is not indispensable for p21 and Mdm-2 mRNA induction in the established p53-cascade. On the other hand, in a reconstituted reaction system, purified PARP from human placenta suppressed the pRB-phosphorylation activity in the presence of NAD and damaged DNA. Human PARP expressed in E. coli showed a similar effect on pRB-phosphorylation activity of cdk2. These findings suggest a direct involvement of PARP in the regulation of cdk activity for cell-cycle arrest.
Mol Cell Biochem 1999 Mar
PMID:Function of poly(ADP-ribose) polymerase in response to DNA damage: gene-disruption study in mice. 1033 51


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