Cloning and expression of cDNA for human poly(ADP-ribose) polymerase.

Alkhatib, Hussein; Chen, Defeng; Cherney, Barry; Bhatia, Kishor; Notario, Vicente; Giri, Chandrakant P.; Stein, Gary S.; Slattery, Elizabeth; Roeder, Robert G.; Smulson, Mark E.

doi:10.1073/pnas.84.5.1224

Cited by 83 publications

(45 citation statements)

References 17 publications

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“…PARP7/7 ®broblasts were transfected with the use of lipofectamine (Life Technologies) with a plasmid encoding wild-type human PARP (pCD12; (Alkhatib et al, 1987)) and the plasmid pTracer-CMV (Invitrogen), a zeocin-based vector system. This vector system was used because the PARP7/7 ®broblasts express an endogenous neomycin resistance gene as a result of the procedure used to establish the original PARP knockout mice.…”

Section: Cells Vectors and Transfectionmentioning

confidence: 99%

Poly(ADP-ribose) polymerase upregulates E2F-1 promoter activity and DNA pol α expression during early S phase

et al. 1999

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E2F-1, a transcription factor implicated in the activation of genes required for S phase such as DNA pol a, is regulated by interactions with Rb and by cell-cycle dependent alterations in E2F-1 abundance. We have shown that depletion of poly(ADP-ribose) polymerase (PARP) by antisense RNA expression downregulates pol a and E2F-1 expression during early S phase. To examine the role of PARP in the regulation of pol a and E2F-1 gene expression, we utilized immortalized mouse ®broblasts derived from wild-type and PARP knockout (PARP7/7) mice as well as PARP7/7 cells stably transfected with PARP cDNA [PARP7/7(+PARP)]. After release from serum deprivation, wild-type and PARP7/7(+PARP) cells, but not PARP7/7 cells, exhibited a peak of cells in S phase by 16 h and had progressed through the cell cycle by 22 h. Whereas [ 3 H]thymidine incorporation remained negligible in PARP7/7 cells, in vivo DNA replication maximized after 18 h in wild-type and PARP7/7(+PARP) cells. To investigate the e ect of PARP on E2F-1 promoter activity, a construct containing the E2F-1 gene promoter fused to a luciferase reporter gene was transiently transfected into these cells. E2F-1 promoter activity in control and PARP7/7(+PARP) cells increased eightfold after 9 h, but not in PARP7/7 cells. PARP7/7 cells did not show the marked induction of E2F-1 expression during early S phase apparent in control and PARP7/7(+PARP) cells. RT ± PCR analysis and pol a activity assays revealed the presence of pol a transcripts and a sixfold increase in activity in both wildtype and PARP7/7(+PARP) cells after 20 h, but not in PARP7/7 cells. These results suggest that PARP plays a role in the induction of E2F-1 promoter activity, which then positively regulates both E2F-1 and pol a expression, when quiescent cells reenter the cell cycle upon recovery from aphidicolin exposure or removal of serum.

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Section: Cells Vectors and Transfectionmentioning

confidence: 99%

Poly(ADP-ribose) polymerase upregulates E2F-1 promoter activity and DNA pol α expression during early S phase

et al. 1999

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“…PARP Binding Reactions-A recombinant full-length human PARP (R&D Systems) was used in DNA binding reactions at a 4:1 molar ratio (protein to DNA) under the ionic conditions required for optimal PARP activity (4,21). The heteroduplex DNA (23) containing stable 50-bp hairpin arms was used in PARP binding reactions.…”

Section: Methodsmentioning

confidence: 99%

“…The 5Ј-deletion mutant of the PARP promoter (p⌬PR-PARP) was generated as described previously (20). The expression plasmid pCD12 containing cDNA for human PARP has been described previously (21). pPARP-DBD was constructed by cloning the PCR-generated fragment of cDNA (22) for human PARP-DBD (amino acids 1-303) tagged at its carboxyl terminus with a sequence encoding four FLAG epitope tags into pcDNA 3.1.…”

Section: Methodsmentioning

confidence: 99%

Transcriptional Repression by Binding of Poly(ADP-ribose) Polymerase to Promoter Sequences

Soldatenkov¹,

Chasovskikh²,

Potaman³

et al. 2002

Journal of Biological Chemistry

104

106

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Poly(ADP-ribose) polymerase (PARP) is a DNA-binding enzyme that plays roles in response to DNA damage, apoptosis, and genetic stability. Recent evidence has implicated PARP in transcription of eukaryotic genes. However, the existing paradigm tying PARP function to the presence of DNA strand breaks does not provide a mechanism by which it may be recruited to gene-regulating domains in the absence of DNA damage. Here we report that PARP can bind to the DNA secondary structures (hairpins) in heteroduplex DNA in a DNA end-independent fashion and that automodification of PARP in the presence of NAD ؉ inhibited its hairpin binding activity. Atomic force microscopic images show that in vitro PARP protein has a preference for the promoter region of the PARP gene in superhelical DNA where the dyad symmetry elements likely form hairpins according to DNase probing. Using a chromatin cross-linking and immunoprecipitation assay we show that PARP protein binds to the chromosomal PARP promoter in vivo. Reporter gene assays have revealed that the transcriptional activity of the PARP promoter is 4 -5-fold greater in PARP knockout cells than in wild type fibroblasts. Reintroduction of vectors expressing full-length PARP protein or its truncated mutant (DNA-binding domain retained but lacking catalytic activity) into PARP ؊/؊ cells has conferred transcriptional down-regulation of the PARP gene promoter. These data provide support for PARP protein as a potent regulator of transcription including down-regulation of its own promoter.Poly(ADP-ribose) polymerase (PARP, 1 EC 2.4.2.30) is a chromatin-associated enzyme that catalyzes the transfer of successive units of the ADP-ribose moiety from NAD ϩ to itself and other nuclear acceptor proteins (1). PARP is a zinc fingercontaining protein, which allows enzyme binding to either double or single strand DNA breaks without any apparent sequence preference (2, 3). The catalytic activity of PARP is strictly dependent on the presence of strand breaks in DNA and is modulated by the level of automodification (4,5). Data from many studies show that PARP is involved in numerous biological functions, all of which are associated with breaking and rejoining DNA strands, and it plays a pivotal role in DNA damage repair (2, 6 -8).Recent studies have implicated PARP in transcription of eukaryotic genes (9 -16). PARP-dependent gene regulation involves poly(ADP-ribosyl)ation of transcription factors, which, in turn, prevents their binding to specific promoter sequences (10). The basal transcription factors TFIIF and TEF-1 as well as transcription factors TATA box-binding protein, YY1, SP-1, cAMP-response element-binding protein, p53, and NFB are all highly specific substrates for poly(ADP-ribosyl)ation (10,11,14,16). PARP may also interact directly with gene promoters. For instance, recombinant full-length PARP bound the DNA sequences within the MCAT1 regulatory element (11) and to the DF4 protein binding site of the Pax-6 gene neuroretinaspecific enhancer (17). Furthermore, PARP involvement in the active...

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“…Disruption of cyclin A expression results in cell cycle arrest at G 2 (25), which might explain, at least in part, the accumulation of PARP Ϫ/Ϫ fibroblasts at G 2 -M. PARP or poly(ADP-ribosyl)ation is also thought to play a role at or near the S-G 2 transition. Thus, the abundance of PARP mRNA (26), poly(ADP-ribose) (27), and poly(ADPribose)-linked acceptor proteins such as histone H1 dimers (28) increases markedly at the S-G 2 transition. Furthermore, chemical inhibitors of PARP also arrest cells at G 2 (29).…”

Section: Reduced Expression Of Genes Important In Cell Cycle Regulatimentioning

confidence: 99%

Misregulation of gene expression in primary fibroblasts lacking poly(ADP-ribose) polymerase

Simbulan-Rosenthal

Rosenthal

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

124

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Poly(ADP-ribose) polymerase (PARP) is implicated in the maintenance of genomic integrity, given that inhibition or depletion of this enzyme increases genomic instability in cells exposed to genotoxic agents. We previously showed that immortalized fibroblasts derived from PARP ؊/؊ mice exhibit an unstable tetraploid population, and partial chromosomal gains and losses in PARP ؊/؊ mice and immortalized fibroblasts are accompanied by changes in the expression of p53, Rb, and c-Jun, as well as other proteins. A tetraploid population has also now been detected in primary fibroblasts derived from PARP ؊/؊ mice. Oligonucleotide microarray analysis was applied to characterize more comprehensively the differences in gene expression between asynchronously dividing primary fibroblasts derived from PARP ؊/؊ mice and their wild-type littermates. Of the 11,000 genes monitored, 91 differentially expressed genes were identified. The loss of PARP results in down-regulation of the expression of several genes involved in regulation of cell cycle progression or mitosis, DNA replication, or chromosomal processing or assembly. PARP deficiency also up-regulates genes that encode extracellular matrix or cytoskeletal proteins that are implicated in cancer initiation or progression or in normal or premature aging. These results provide insight into the mechanism by which PARP deficiency impairs mitotic function, thereby resulting in the genomic alterations and chromosomal abnormalities as well as in altered expression of genes that may contribute to genomic instability, cancer, and aging.

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Cloning and expression of cDNA for human poly(ADP-ribose) polymerase.

Cited by 83 publications

References 17 publications

Poly(ADP-ribose) polymerase upregulates E2F-1 promoter activity and DNA pol α expression during early S phase

Poly(ADP-ribose) polymerase upregulates E2F-1 promoter activity and DNA pol α expression during early S phase

Transcriptional Repression by Binding of Poly(ADP-ribose) Polymerase to Promoter Sequences

Misregulation of gene expression in primary fibroblasts lacking poly(ADP-ribose) polymerase

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