Overlap of the gene encoding the novel poly(ADP-ribose) polymerase Parp10 with the plectin 1 gene and common use of exon sequences

We have recently identified PARP10 as a novel functional poly(ADP-ribose) polymerase. The gene encoding PARP10 is conserved in vertebrates but no orthologs were found in lower organisms. In addition to the poly(ADP-ribose) polymerase domain, PARP10 possesses several additional sequence motifs, including an RNA recognition motif and two ubiquitin interaction motifs. We characterized the murine genomic locus of the Parp10 gene. We noticed that 3' Parp10 sequences overlapped with the plectin 1 gene in a head-to-tail arrangement. Detailed analyses revealed that the two most 3' Parp10 exons (exons 10 and 11) are also used for plectin 1. While these two exons code for part of the poly(ADP-ribose) polymerase domain in Parp10, they are noncoding for plectin 1 due to the lack of appropriate start codons. Furthermore our findings suggest that at least one of the plectin 1 promoters is located within intron 9 of the Parp10 gene.     

ADP-ribose polymers localized on Ctcf-Parp1-Dnmt1 complex prevent methylation of Ctcf target sites

PARylation [poly(ADP-ribosyl)ation] is involved in the maintenance of genomic methylation patterns through its control of Dnmt1 [DNA (cytosine-5)-methyltransferase 1] activity. Our previous findings indicated that Ctcf (CCCTC-binding factor) may be an important player in key events whereby PARylation controls the unmethylated status of some CpG-rich regions. Ctcf is able to activate Parp1 [poly(ADP-ribose) polymerase 1], which ADP-ribosylates itself and, in turn, inhibits DNA methylation via non-covalent interaction between its ADP-ribose polymers and Dnmt1. By such a mechanism, Ctcf may preserve the epigenetic pattern at promoters of important housekeeping genes. The results of the present study showed Dnmt1 as a new protein partner of Ctcf. Moreover, we show that Ctcf forms a complex with Dnmt1 and PARylated Parp1 at specific Ctcf target sequences and that PARylation is responsible for the maintenance of the unmethylated status of some Ctcf-bound CpGs. We suggest a mechanism by which Parp1, tethered and activated at specific DNA target sites by Ctcf, preserves their methylation-free status.     

The response of Parp knockout mice against DNA damaging agents

Gene-disruption studies involving poly(ADP-ribose) polymerase (Parp) have identified the various roles of Parp in cellular responses to DNA damage. The partial rescue of V[D]J recombination process in SCID/Parp(-/-) double mutant mice indicates the participation of Parp in the repair of DNA strand break. Parp(-/-) mice are more sensitive to the lethal effects of alkylating agents. Parp is also thought to be involved in base-excision repair after DNA damage caused by alkylating agents. On the other hand, resistance of Parp(-/-) mice to DNA damage induced by reactive oxygen species implicates the contribution of Parp to cell death through NAD depletion. Parp(-/-) mice with two different genetic backgrounds also show enhanced sensitivity to the lethal effects of gamma-irradiation. Parp(-/-) mice show more severe villous atrophy of the small intestine compared to the wild-type counterpart in a genetic background of 129Sv/C57BL6. Other forms of enhanced tissue damage have been identified in Parp(-/-) mice with a genetic background of 129Sv/ICR. For example, Parp(-/-) mice exhibit extensive hemorrhage in the glandular stomach and other tissues, such as the testes, after gamma-irradiation. Severe myelosuppression is also observed in both Parp(+/+) and Parp(-/-) mice, but Parp(+/+) mice show extensive extramedullary hematopoiesis in the spleen during the recovery phase of post-irradiation, whereas the spleen of Parp(-/-) mice exhibits severe atrophy with no extramedullary hematopoiesis. The absence of extramedullary hematopoiesis in the spleen is probably the underlying mechanism of hemorrhagic tendency in various tissues of Parp(-/-) mice. These findings suggest that loss of Parp activity could contribute to post-irradiation tissue hemorrhage.     

Parp1-deficiency induces differentiation of ES cells into trophoblast derivatives

Embryonic stem (ES) cells deficient in the enzyme poly(ADP-ribose) polymerase (Parp1) develop into teratocarcinoma-like tumors when injected subcutaneously into nude mice that contain cells with giant cell-like morphology. We show here that these cells express genes characteristic of trophoblast giant cells and thus belong to the trophectoderm lineage. In addition, Parp1(-/-) tumors contained other trophoblast subtypes as revealed by expression of spongiotrophoblast-specific marker genes. The extent of giant cell differentiation was enhanced, however, as compared with spongiotrophoblast. A similar shift toward trophoblast giant cell differentiation was observed in cultures of Parp1-deficient ES cells and in placentae of Parp1(-/-) embryos. Analysis of other cell lineage markers demonstrated that Parp1 acts exclusively in trophoblast to suppress differentiation. Surprisingly, trophoblast derivatives were also detected in wildtype tumors and cultured ES cells, albeit at significantly lower frequency. These data show that wildtype ES cells contain a small population of cells with trophectoderm potential and that absence of Parp1 renders ES cells more susceptible to adopting a trophoblast phenotype.     

Growth-phase-dependent response to DNA damage in poly(ADP-ribose) polymerase deficient cell lines: basis for a new hypothesis describing the role of poly(ADP-ribose) polymerase in DNA replication and repair

We have studied the role of poly(ADP-ribose) polymerase in the repair of DNA damage induced by x-ray and N-methyl N-nitro-N-nitrosoguanidine (MNNG) by using V79 chinese hamster cells, and two derivative mutant cell lines, ADPRT54 and ADPRT351, that are deficient in poly(ADP-ribose) polymerase activity. Under exponentially growing conditions these mutant cell lines are hypersensitive to x-irradiation and MNNG compared to their parental V79 cells which could be interpreted to suggest that poly(ADP-ribose) polymerase is involved in the repair of DNA damage. However, the level of DNA strand breaks induced by x-irradiation and MNNG and their rates of repair are similar in all the cell lines, thus suggesting that it may not be the difference in strand break formation or in its rate of repair that is contributing to the enhanced cell killing in exponentially growing poly(ADP-ribose) polymerase deficient cell lines. In contrast, under growth-arrested conditions, all three cell lines become similarly sensitive to both x-irradiation and MNNG, thus suggesting that poly(ADP-ribose) polymerase may not be involved in the repair of DNA damage in growth-arrested cells. These paradoxical results could be interpreted to suggest that poly(ADP-ribose) polymerase is involved in DNA repair in a cell-cycle-dependent fashion, however, it is functionally active throughout the cell cycle. To resolve this dilemma and explain these results and those obtained by many others, we propose that the normal function of poly(ADP-ribose) polymerase is to prevent DNA recombination processes and facilitate DNA ligation.     

Characterization of human poly(ADP-ribose) polymerase with autoantibodies

The addition of poly(ADP-ribose) chains to nuclear proteins has been reported to affect DNA repair and DNA synthesis in mammalian cells. The enzyme that mediates this reaction, poly(ADP-ribose) polymerase, requires DNA for catalytic activity and is activated by DNA with strand breaks. Because the catalytic activity of poly(ADP-ribose) polymerase does not necessarily reflect enzyme quantity, little is known about the total cellular poly(ADP-ribose) polymerase content and the rate of its synthesis and degradation. In the present experiments, specific human autoantibodies to poly(ADP-ribose) polymerase and a sensitive immunoblotting technique were used to determine the cellular content of poly(ADP-ribose) polymerase in human lymphocytes. Resting peripheral blood lymphocytes contained 0.5 X 10(6) enzyme copies per cell. After stimulation of the cells by phytohemagglutinin, the poly(ADP-ribose) polymerase content increased before DNA synthesis. During balanced growth, the T lymphoblastoid cell line CEM contained approximately 2 X 10(6) poly(ADP-ribose) polymerase molecules per cell. This value did not vary by more than 2-fold during the cell growth cycle. Similarly, mRNA encoding poly(ADP-ribose) polymerase was detectable throughout S phase. Poly(ADP-ribose) polymerase turned over at a rate equivalent to the average of total cellular proteins. Neither the cellular content nor the turnover rate of poly(ADP-ribose) polymerase changed after the introduction of DNA strand breaks by gamma irradiation. These results show that in lymphoblasts poly(ADP-ribose) polymerase is an abundant nuclear protein that turns over relatively slowly and suggest that most of the enzyme may exist in a catalytically inactive state.