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The Arabidopsis thaliana Poly(ADP-Ribose) Polymerases 1 and 2 Modify DNA by ADP-Ribosylating Terminal Phosphate Residues.

ABSTRACT: Proteins from the poly(ADP-ribose) polymerase (PARP) family, such as PARP1 and PARP2, use NAD⁺ as a substrate to catalyze the synthesis of polymeric chains consisting of ADP-ribose units covalently attached to an acceptor molecule. PARP1 and PARP2 are viewed as DNA damage sensors that, upon binding to strand breaks, poly(ADP-ribosyl)ate themselves and nuclear acceptor proteins. The flowering plant Arabidopsis thaliana contains three genes encoding homologs of mammalian PARPs: atPARP1, atPARP2, and atPARP3. Both atPARP1 and atPARP2 contain poly(ADP-ribosyl)ating activity; however, it is unknown whether they could covalently modify DNA by ADP-ribosylating the strand break termini. Here, we report that similar to their mammalian counterparts, the plant atPARP1 and atPARP2 proteins ADP-ribosylate 5'-terminal phosphate residues in duplex DNA oligonucleotides and plasmid containing at least two closely spaced DNA strand breaks. AtPARP1 preferentially catalyzes covalent attachment of ADP-ribose units to the ends of recessed DNA duplexes containing 5'-phosphate, whereas atPARP2 preferentially ADP-ribosylates the nicked and gapped DNA duplexes containing the terminal 5'-phosphate. Similar to their mammalian counterparts, the plant PARP-catalyzed DNA ADP-ribosylation is particularly sensitive to the distance that separates two strand breaks in the same DNA molecule, 1.5 and 1 or 2 turns of helix for atPARP1 and atPARP2, respectively. PAR glycohydrolase (PARG) restored native DNA structure by hydrolyzing the PAR-DNA adducts generated by atPARPs. Biochemical and mass spectrometry analyses of the PAR-DNA adducts showed that atPARPs utilize phosphorylated DNA termini as an alternative to protein acceptor residues to catalyze PAR chain synthesis via phosphodiester bond formation between C1' of ADP-ribose and a phosphate residue of the terminal nucleotide in DNA fragment. Taken together, these data establish the presence of a new type of DNA-modifying activity in Arabidopsis PARPs, suggesting a possible role of DNA ADP-ribosylation in DNA damage signaling and repair of terrestrial plants.

SUBMITTER: Taipakova S

PROVIDER: S-EPMC7726343 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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The Arabidopsis thaliana Poly(ADP-Ribose) Polymerases 1 and 2 Modify DNA by ADP-Ribosylating Terminal Phosphate Residues.

Taipakova Sabira S Kuanbay Aigerim A Saint-Pierre Christine C Gasparutto Didier D Baiken Yeldar Y Groisman Regina R Ishchenko Alexander A AA Saparbaev Murat M Bissenbaev Amangeldy K AK

Frontiers in cell and developmental biology 20201126

Proteins from the poly(ADP-ribose) polymerase (PARP) family, such as PARP1 and PARP2, use NAD+ as a substrate to catalyze the synthesis of polymeric chains consisting of ADP-ribose units covalently attached to an acceptor molecule. PARP1 and PARP2 are viewed as DNA damage sensors that, upon binding to strand breaks, poly(ADP-ribosyl)ate themselves and nuclear acceptor proteins. The flowering plant Arabidopsis thaliana contains three genes encoding homologs of mammalian PARPs: < ...[more]

PMID: 33324653

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Project description:BackgroundInitiation, amplitude, duration and termination of transforming growth factor ? (TGF?) signaling via Smad proteins is regulated by post-translational modifications, including phosphorylation, ubiquitination and acetylation. We previously reported that ADP-ribosylation of Smads by poly(ADP-ribose) polymerase 1 (PARP-1) negatively influences Smad-mediated transcription. PARP-1 is known to functionally interact with PARP-2 in the nucleus and the enzyme poly(ADP-ribose) glycohydrolase (PARG) can remove poly(ADP-ribose) chains from target proteins. Here we aimed at analyzing possible cooperation between PARP-1, PARP-2 and PARG in regulation of TGF? signaling.MethodsA robust cell model of TGF? signaling, i.e. human HaCaT keratinocytes, was used. Endogenous Smad3 ADP-ribosylation and protein complexes between Smads and PARPs were studied using proximity ligation assays and co-immunoprecipitation assays, which were complemented by in vitro ADP-ribosylation assays using recombinant proteins. Real-time RT-PCR analysis of mRNA levels and promoter-reporter assays provided quantitative analysis of gene expression in response to TGF? stimulation and after genetic perturbations of PARP-1/-2 and PARG based on RNA interference.ResultsTGF? signaling rapidly induces nuclear ADP-ribosylation of Smad3 that coincides with a relative enhancement of nuclear complexes of Smads with PARP-1 and PARP-2. Inversely, PARG interacts with Smads and can de-ADP-ribosylate Smad3 in vitro. PARP-1 and PARP-2 also form complexes with each other, and Smads interact and activate auto-ADP-ribosylation of both PARP-1 and PARP-2. PARP-2, similar to PARP-1, negatively regulates specific TGF? target genes (fibronectin, Smad7) and Smad transcriptional responses, and PARG positively regulates these genes. Accordingly, inhibition of TGF?-mediated transcription caused by silencing endogenous PARG expression could be relieved after simultaneous depletion of PARP-1.ConclusionNuclear Smad function is negatively regulated by PARP-1 that is assisted by PARP-2 and positively regulated by PARG during the course of TGF? signaling.

Project description:Abiotic and biotic stress can have a detrimental impact on plant growth and productivity. Hence, there is a substantial demand for key factors of stress responses to improve yield stability of crops. Members of the poly(ADP-ribose)polymerase (PARP) protein family, which post-translationally modify (PARylate) nuclear proteins, have been suggested as such universal determinants of plant stress responses. A role under abiotic stress has been inferred from studies in which a genetic or, more commonly, pharmacological inhibition of PARP activity improved the performance of stressed plants. To further elucidate the role of PARP proteins under stress, T-DNA knockout mutants for the three Arabidopsis thaliana PARP genes were subjected to drought, osmotic, salt, and oxidative stress. To exclude a functional redundancy, which was indicated by a transcriptional upregulation of the remaining parp genes, a parp triple mutant was generated. Surprisingly, parp mutant plants did not differ from wild type plants in any of these stress experiments, independent from the number of PARP genes mutated. The parp triple mutant was also analyzed for callose formation in response to the pathogenassociated molecular pattern flg22. Unexpectedly, callose formation was unaltered in the mutant, albeit pharmacological PARP inhibition robustly blocked this immune response, confirming previous reports. Evidently, pharmacological inhibition appears to be more robust than the abolition of all PARP genes, indicating the presence of so-far undescribed proteins with PARP activity. This was supported by the finding that protein PARylation was not absent, but even increased in the parp triple mutant. Candidates for novel PARP-inhibitor targets may be found in the SRO protein family. These proteins harbor a catalytic PARP-like domain and are centrally involved in stress responses. Molecular modeling analyses, employing animal PARPs as templates, indeed indicated a capability of the SRO proteins RCD1 and SRO1 to bind nicotinamide-derived inhibitors. Collectively, the results of our study suggest that the stress-related phenotypes of parp mutants are highly conditional, and they call for a reconsideration of PARP inhibitor studies. In the context of this study, we also propose a unifying nomenclature of PARP genes and parp mutants, which is currently highly inconsistent and redundant.

Dataset Information

The Arabidopsis thaliana Poly(ADP-Ribose) Polymerases 1 and 2 Modify DNA by ADP-Ribosylating Terminal Phosphate Residues.

Publications

The <i>Arabidopsis thaliana</i> Poly(ADP-Ribose) Polymerases 1 and 2 Modify DNA by ADP-Ribosylating Terminal Phosphate Residues.

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