PARG is recruited to DNA damage sites through poly(ADP-ribose)- and PCNA-dependent mechanisms

Post-translational poly(ADP-ribosyl)ation has diverse essential functions in the cellular response to DNA damage as it contributes to avid DNA damage detection and assembly of the cellular repair machinery but extensive modification eventually also induces cell death. While there are 17 human poly(ADP-ribose) polymerase (PARP) genes, there is only one poly(ADP-ribose) glycohydrolase (PARG) gene encoding several PARG isoforms located in different subcellular compartments. To investigate the recruitment of PARG isoforms to DNA repair sites we locally introduced DNA damage by laser microirradiation. All PARG isoforms were recruited to DNA damage sites except for a mitochondrial localized PARG fragment. Using PARP knock out cells and PARP inhibitors, we showed that PARG recruitment was only partially dependent on PARP-1 and PAR synthesis, indicating a second, PAR-independent recruitment mechanism. We found that PARG interacts with PCNA, mapped a PCNA binding site and showed that binding to PCNA contributes to PARG recruitment to DNA damage sites. This dual recruitment mode of the only nuclear PARG via the versatile loading platform PCNA and by a PAR dependent mechanism likely contributes to the dynamic regulation of this posttranslational modification and ensures the tight control of the switch between efficient DNA repair and cell death

Feedback-regulated poly(ADP-ribosyl)ation by PARP-1 is required for rapid response to DNA damage in living cells

Genome integrity is constantly threatened by DNA lesions arising from numerous exogenous and endogenous sources. Survival depends on immediate recognition of these lesions and rapid recruitment of repair factors. Using laser microirradiation and live cell microscopy we found that the DNA-damage dependent poly(ADP-ribose) polymerases (PARP) PARP-1 and PARP-2 are recruited to DNA damage sites, however, with different kinetics and roles. With specific PARP inhibitors and mutations, we could show that the initial recruitment of PARP-1 is mediated by the DNA-binding domain. PARP-1 activation and localized poly(ADP-ribose) synthesis then generates binding sites for a second wave of PARP-1 recruitment and for the rapid accumulation of the loading platform XRCC1 at repair sites. Further PARP-1 poly(ADP-ribosyl)ation eventually initiates the release of PARP-1. We conclude that feedback regulated recruitment of PARP-1 and concomitant local poly(ADP-ribosyl)ation at DNA lesions amplifies a signal for rapid recruitment of repair factors enabling efficient restoration of genome integrity

Selective modulation by PARP-1 of HIF-1α-recruitment to chromatin during hypoxia is required for tumor adaptation to hypoxic conditions

[Background] The adaptation to hypoxia is mainly controlled by the HIF transcription factors. Increased expression/activity of HIF-1α correlates with poor prognosis in cancer patients. PARP-1 inhibitors are used in the clinic to treat BRCAness breast/ovarian cancer and have been shown to regulate the hypoxic response; therefore, their use could be expanded.[Methods] In this work by integrating molecular/cell biology approaches, genome-wide ChIP-seq, and patient samples, we elucidate the extent to which PARP-1 exerts control over HIF-1-regulated genes.[Results] In human melanoma, PARP-1 and HIF-1α expression are strongly associated. In response to a hypoxic challenge poly(ADP-ribose) (PAR) is synthesized, HIF-1α is post-transcriptionally modified (PTM) and stabilized by PARylation at specific K/R residues located at its C-terminus. Using an unbiased ChIP-seq approach we demonstrate that PARP-1 dictates hypoxia-dependent HIF-recruitment to chromatin in a range of HIF-regulated genes while analysis of HIF-binding motifs (RCGTG) reveals a restriction on the recognition of hypoxia responsive elements in the absence of PARP-1. Consequently, the cells are poorly adapted to hypoxia, showing a reduced fitness during hypoxic induction.[Conclusions] These data characterize the fine-tuning regulation by PARP-1/PARylation of HIF activation and suggest that PARP inhibitors might have therapeutic potential against cancer types displaying HIF-1α over-activation.This work was supported by Junta de Andalucía, project of Excellence from Junta de Andalucía P10-CTS-0662, P12-CTS-383 to FJO, Spanish Ministry of Economy and Competitiveness SAF2012-40011-C02-01, SAF2015-70520- R, RTI2018-098968-B-I00, RTICC RD12/0036/0026 and CIBER Cáncer ISCIII CB16/12/00421 to FJO. EB1s lab is supported by the Basque Department of Industry, Tourism and Trade (Etortek) and the MINECO (CB16/12/00421) grants. Fundación Domingo Martínez (call 2019).Peer reviewe

Etude des mécanismes de maturation des précurseurs de tRNA dans la mitochondrie de levure: RNase P et 3’ pré-tRNase mitochondriales

This thesis concerns the study of the enzymatic activities of the mitochondria of the yeast Saccharomyces cerevisiae, responsible for the 5 'and 3' maturation of the tRNAs carried out by RNase P and 3' pre-tRNase, respectively.Most of this work is devoted to the purification of mitochondrial yeast RNase P and the study of its ribonucleic and protein constituents. Then, as part of a study of the structure-function relationships of the ribonucleic component of mitochondrial RNase P, we identified the latter in the mitochondria of various yeasts of the genus Kluyveromyces, evolving very close to the genus Saccharomyces. Finally, we characterized the 3 'mitochondrial pre-tRNase and studied some of its properties.RNase P is a ribonucleoprotein consisting of RNA 9S encoded by a mitochondrial gene (RPM1 gene) and core encoded protein (s). The enzyme has been purified to apparent homogeneity. We have demonstrated that 9S RNA is the ribonucleic component of RNase P. However, "Northern" hybridization experiments on RNA extracted at different stages of enzyme purification, using probes from different regions of the gene. RPM1, showed that the 9S RNA was degraded during the purification, without significantly affecting the activity of the enzyme. On the other hand, the extraction of RNA from the purified RNase P made it possible to isolate an RNA of 70 nucleotides whose sequence corresponds to that of the 3 'end of the 9S RNA. The presence of this fragment in the ribonucleoprotein appears to be sufficient for its in vitro activity. A 63 kDa polypeptide co-purifies with RNase P activity. It has a native gel mass of 250 kDa, suggesting that it could be composed of 4 subunits of the 63 kDa polypeptide and an RNA fragment. 9S of 70 nucl. The 63 kDa polypeptide is revealed with sera from patients with autoimmune diseases. These serum directed against ribonucleoproteins, are in particular capable of recognizing the RNase P of E. coli and Hela cells. Thus, the protein component of RNAse P could have structural homologies with other ribonucleoproteins recognized by this family of antibodies, such as U1-snRNP and thRNP (RNase MRP).To clarify the structure-function relationships of the yeast mitochondrial RNase P RNA, we identified genes equivalent to that of 9S RNA in mitochondria of different species of the genus Kluyveromyces (K. bulgaricus, K. fragilis, K. thermotolerans). ) and S. carlsbergensis, evolutionarily close to the genus Saccharomyces. The RNA gene of these four species was cloned by PCR. We have demonstrated that this gene actually codes for the ribonucleic component of RNase P. Indeed, the presence of RNA correlates perfectly with the enzymatic activity of RNase P in partially purified mitochondrial fractions. The size and sequence of these RNAs were highly variable from one species to another. The only elements of conserved sequences are located in two short regions, one at the 5 'end and the other at the 3' end of the molecule. These two blocks, which represent the only elements of sequences conserved between the RNA of eubacteria and that of yeast mitochondria, are capable of pairing together to form a "pseudo-node" structure. This tertiary folding could be crucial for the function of these RNAs. The sequence alignment of these mitochondrial RNAs allowed us to propose a common model of secondary structure, comparable to that of RNAse P prokaryotic RNAs.We have characterized the endonuclease activity involved in the maturation of the 3' end of mitochondrial tRNAs. Unlike mitochondrial RNase P, the activity of this enzyme does not depend on the presence of a ribonucleic acid. Kinetic studies of maturation, carried out with a tRNA precursor possessing both 5 'and 3' extensions, made it possible to demonstrate the existence of a chronology in the maturation of mitochodrial tRNAs. Indeed, in protein fractions containing both the activity of RNase P and that of 3' pre-tRNase, the 5' ends are removed first, then in a second step, the 3 'pre-tRNase cleaves. the extension 3' of the precursor. Unfortunately, the purification of the 3' pre-tRNase has stumbled upon a problem of stability of this enzyme which has therefore not been studied in greater detail.Ce mémoire porte sur l’étude des activités enzymatiques de la mitochondrie de la levure Saccharomyces cerevisiae, responsables de la maturation en 5’ et en 3’ des tRNA réalisée par la RNase P et la 3’ pré-tRNase, respectivement.La majeure partie de ce travail est consacrée à la purification de la RNase P mitochondriale de levure et à l’étude de ses constituants ribonucléique et protéique. Puis, dans le cadre d’une étude des relations structure-fonctions du composant ribonucléique de la RNase P mitochondriale, nous avons identifié ce dernier dans les mitochondries de différentes levures du genre Kluyveromyces, évolutivement très proches du genre Saccharomyces. Enfin, nous avons caractérisé la 3’ pré-tRNase mitochondriale et étudié quelques-unes de ses propriétés.La RNase P est une ribonucléoprotéine constituée du RNA 9S codé par un gène mitochondriale (gène RPM1) et de protéine(s) codée(s) par le noyau. L’enzyme a été purifié à une apparente homogénéité. Nous avons démontré que le RNA 9S est le composant ribonucléique de la RNase P. Cependant, des expériences d’hybridation “Northern” sur le RNA extrait à différentes étapes de la purification de l’enzyme, en utilisant des sondes de différentes régions du gène RPM1, ont montré que le RNA 9S était dégradé au cours de la purification, sans pour autant affecter de façon significative l’activité de l’enzyme. D’autre part, l’extraction du RNA à partir de la RNase P purifiée a permis d’isoler un RNA de 70 nucléotides dont la séquence correspond à celle de l’extrémité 3’ du RNA 9S. La présence de ce fragment dans la ribonucléoprotéine semble être suffisante pour son activité in vitro. Un polypeptide de 63 kDa co-purifie avec l’activité RNase P. Il a une masse de 250 kDa sur gel natif, suggérant qu’il pourrait être composé de 4 sous-unités du polypeptide de 63 kDa et d’un fragment du RNA 9S de 70 nucl. Le polypeptide de 63 kDa est révélé avec des séra de malades atteints de maladies auto-immunes. Ces séra dirigés contre des ribonucléoprotéines, sont notamment capables de reconnaˆıtre la RNase P de E. coli et de cellules Hela. Ainsi, le composant protéique de la RNAse P pourrait avoir des homologies structurales avec d’autres ribonucléoprotéines reconnues par cette famille d’anticorps, comme par exemple la U1-snRNP et la thRNP(RNase MRP).Afin de préciser les relations structure-fonctions du RNA de la RNase P mitochondriale de levure, nous avons identifié des gènes équivalents à celui du RNA 9S dans les mitochondries de différentes espèces du genre Kluyveromyces (K. bulgaricus, K. fragilis, K. thermotolerans) et de S. carlsbergensis, évolutivement proches du genre Saccharomyces. Le gène de ce RNA de ces quatre espèces a été cloné par PCR. Nous avons démontré que ce gène code effectivement pour le constituant ribonucléique de la RNase P. En effet, la présence du RNA corrèle parfaitement avec l’activité enzymatique de la RNase P dans des fractions mitochondriales partiellement purifiées. La taille et la séquence de ces RNA se sont révélées très variables d’une espèce à l’autre. Les seuls éléments de séquences conservées sont localisés dans deux courtes régions situées, l’une à l’extrémité 5’ et l’autre à l’extrémité 3’ de la molécule. Ces deux blocs qui représentent les seuls éléments de séquences conservées entre le RNA des eubactéries et celui des mitochondries de levures, sont capables de s’apparier pour former une structure en “pseudo-nœud”. Ce repliement tertiaire pourrait être crucial pour la fonction de ces RNA. L’alignement des séquences de ces RNA mitochondriaux nous a permis de proposer un modèle commun de structure secondaire, comparable à celui des RNA des RNAse P procaryotes.Nous avons caractérisé l’activité endonucléasique impliquée dans la maturation de l’extrémité 3’ des tRNA mitochondriaux. Contrairement à la RNase P mitochondriale, l’activité de cet enzyme ne dépend pas de la présence d’un acide ribonucléique. Des études cinétiques de maturation, réalisées avec un précurseur de tRNA possédant les deux extensions en 5’ et en 3’, ont permis de mettre en évidence l’existence d’une chronologie dans la maturation des tRNA mitochodriaux. En effet, dans des fractions protéiques contenant à la fois l’activité de la RNase P et celle de la 3’ pré-tRNase, les extrémités 5’ sont enlevées en premier, puis dans un deuxième temps, la 3’ pré-tRNase clive l’extension 3’ du précurseur. Malheureusement, la purification de la 3’ pré-tRNase a buté sur un problème de stabilité de cet enzyme qui n’a donc pu être étudié de façon plus approfondie

An unusually compact external promoter for RNA polymerase III transcription of the human H1RNA gene

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Molecular heterogeneity and regulation of poly(ADP-ribose) glycohydrolase.

International audienceWe 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

The PARP superfamily

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Purification of Recombinant Human PARP-3

International audienceThe purification of poly(ADP-ribose) polymerase-3 (PARP-3) from overexpressing cells (Sf9 insect cells, Escherichia coli) has been updated to a fast and reproducible two chromatographic steps protocol. After cell lysis, PARP-3 protein from the crude extract is affinity purified on a 3-aminobenzamide Sepharose™ chromatographic step. The last contaminants and the 3-methoxybenzamide used to elute PARP-3 from the previous affinity column are removed on the high-performance strong cations exchanger MonoQ™ matrix. This step allows also the concentration of the protein. The columns connected to an ÅKTA™ purifier system allow the purification of the protein in 3 days with a high-yield recovery. As described in the protocol, more than 3 mg of pure and active human PARP-3 can be obtained from 1.5 L of E. coli culture