Importance of poly(ADP-ribose) polymerases in the regulation of DNA-dependent processes.

Poly(ADP-ribosyl)ation of proteins is involved in the regulation of basal cellular processes and seems to be crucial for genomic integrity and cell survival. Several nuclear poly(ADP-ribose) polymerases (PARPs) are known which interact with various proteins involved in DNA metabolism. These proteins can be targets of poly(ADP-ribosyl)ation, which generally downregulates their activities. Accordingly, PARPs have been implicated in numerous processes involving chromosomal DNA, such as the regulation of chromatin structure, DNA repair, replication and transcription. PARP-1, the major cellular PARP, and PARP-2 are activated by DNA strand breaks. These enzymes have been shown to participate in DNA repair. PARP-1 has also been associated with DNA replication and recombination. Another outstanding feature of PARP-1 is its impact on the activities of transcription factors and on gene expression. Two other nuclear PARP enzymes, tankyrase-1 and tankyrase-2, are important for telomere maintenance.

Dephosphorylation of histone gamma-H2AX during repair of DNA double-strand breaks in mammalian cells and its inhibition by calyculin A.

The induction of DNA double-strand breaks (DSBs) by ionizing radiation in mammalian chromosomes leads to the phosphorylation of Ser-139 in the replacement histone H2AX, but the molecular mechanism(s) of the elimination of phosphorylated H2AX (called gamma-H2AX) from chromatin in the course of DSB repair remains unknown. We showed earlier that gamma-H2AX cannot be replaced by exchange with free H2AX, suggesting the direct dephosphorylation of H2AX in chromatin by a protein phosphatase. Here we studied the dynamics of dephosphorylation of gamma-H2AX in vivo and found that more than 50% was dephosphorylated in 3 h, but a significant amount of gamma-H2AX could be detected even 6 h after the induction of DSBs. At this time, a significant fraction of the gamma-H2AX nuclear foci co-localized with the foci of RAD50 protein that did not co-localize with replication sites. However, gamma-H2AX could be detected in some cells treated with methyl methanesulfonate which accumulated RAD18 protein at stalled replication sites. We also found that calyculin A inhibited early elimination of gamma-H2AX and DSB rejoining in vivo and that protein phosphatase 1 was able to remove phosphate groups from gamma-H2AX-containing chromatin in vitro. Our results confirm the tight association between DSBs and gamma-H2AX and the coupling of its in situ dephosphorylation to DSB repair.

[Recognition of internal (TTAGGG)n repeats by telomeric protein TRF1 and its role in maintenance of chromosomal stability in Chinese hamster cells]. / Uznavanie vnutrennikh povtorov (TTAGGG)n telomernym belkom TRF1 i ego rol' v podderzhanii stabil'nosti khromosom v kletkakh kitaiskogo khomiachka.

Mammalian telomeres contain long tandem (TTAGGG)n repeats, which are protected by a complex of different proteins. Telomeric repeat-binding factors TRF1 and TRF2 play the key role in protection of telomeres through the formation of terminal loops (called T-loop). A T-loop isolates the 3' strand telomeric end and with this mechanism protects telomeres from the influence of enzymes of DNA reparation and telomere fusions and also interferes with the interaction of telomerase with telomeres. Many vertebrate species also contain large blocks of (TTAGGG)n sequences in pericentric and interstitial chromosome bands. The Chinese hamster genome contains a total of 18 arrays of these non-telomeric internal (TTAGGG)n sequences (ITs). Chromosome bands containing these arrays are unstable and should be protected with the help of another mechanism, rather than that using telomeres. In this study we analysed association of Green Fluorescent Protein (GFP)-tagged TRF1 in Chinese hamster V79 cells with ITs. We found that in these cells GFP-TRF1 associates with ITs in the interphase nucleus. We detected a little overlap between IT-associated GFP-TRF1 and random DSB sites visualized after the treatment of V79 cells with ionizing radiation. We found that the treatment of V79 cells with WM significantly increases the frequency of spontaneous chromosome aberrations. These WM effects are possible due to inhibiting phosphorylation of TRF1 by ATM. TRF1 is known to be eliminated from telomeres by overexpression of TANK1, which induces TRF1 poly(ADP-ribosyl)ation. We transfected V79 cells by plasmid encoding TANK1 and found that the frequency of chromosome rearrangements increased in these cells independently of their treatment by IR. Taken together, our results suggest that TRF1 may be involved in the sequence-specific protection of internal non-telomeric (TTAGGG)n repeats.

Poly(ADP-ribosyl)ation of transcription factor Yin Yang 1 under conditions of DNA damage.

Under conditions of severe DNA damage the nuclear enzyme poly(ADP-ribose) polymerase 1 (PARP-1) is activated, catalyzing the modification of proteins by forming and attaching to them poly(ADP-ribose) chains. A specific physical interaction between PARP-1 and transcription factor Yin Yang 1 (YY1) in vitro was shown previously, which had important consequences for the activities of both proteins. It is demonstrated here that YY1 and PARP-1 form complexes in vivo. YY1 was transiently poly(ADP-ribosyl)ated immediately after genotoxic treatment of HeLa cells. The narrow time frame of the modification coincides with that known for the activation of PARP-1 under these conditions. This immediate modification correlated with a decreased affinity of YY1 to its cognate DNA binding sites.

Transcription factor Yin Yang 1 stimulates poly(ADP-ribosyl)ation and DNA repair.

Poly(ADP-ribose) polymerase 1 (PARP-1) is a nuclear enzyme that catalyzes the synthesis of ADP-ribose polymers from NAD(+). The function of PARP-1 is related to important nuclear processes including DNA repair and transcription. Previous studies demonstrated a specific physical interaction between PARP-1 and the transcription factor Yin Yang 1 (YY1) in vitro. In this study, a functional relationship between both proteins in response to genotoxic treatment of cells is presented. The interaction of YY1 with PARP-1 greatly stimulates the enzymatic activity of PARP-1. Consistent with this, the overexpression of YY1 in HeLa cells resulted in an enhanced synthesis of poly(ADP-ribose) and an acceleration of DNA repair in response to a treatment with methyl-N'-nitro-N'-nitrosoguanidine.

A cellular survival switch: poly(ADP-ribosyl)ation stimulates DNA repair and silences transcription.

Poly(ADP-ribosyl)ation is a post-translational modification occurring in the nucleus. The most abundant and best-characterized enzyme catalyzing this reaction, poly(ADP-ribose) polymerase 1 (PARP1), participates in fundamental nuclear events. The enzyme functions as molecular "nick sensor". It binds with high affinity to DNA single-strand breaks resulting in the initiation of its catalytic activity. Activated PARP1 promotes base excision repair. In addition, PARP1 modifies several transcription factors and thereby precludes their binding to DNA. We propose that a major function of PARP1 includes the silencing of transcription preventing expression of damaged genes. Concomitant stimulation of DNA repair suggests that PARP1 acts as a switch between transcription and DNA repair. Another PARP-type enzyme, tankyrase, is involved in the regulation of telomere elongation. Tankyrase modifies a telomere-associated protein and thereby prevents it masking telomeric repeats providing access of telomerase for telomere elongation. Therefore, poly(ADP-ribosyl)ation reactions may act as molecular switches in DNA metabolism.

Characterization of recombinant human nicotinamide mononucleotide adenylyl transferase (NMNAT), a nuclear enzyme essential for NAD synthesis.

Nicotinamide mononucleotide adenylyl transferase (NMNAT) is an essential enzyme in all organisms, because it catalyzes a key step of NAD synthesis. However, little is known about the structure and regulation of this enzyme. In this study we established the primary structure of human NMNAT. The human sequence represents the first report of the primary structure of this enzyme for an organism higher than yeast. The enzyme was purified from human placenta and internal peptide sequences determined. Analysis of human DNA sequence data then permitted the cloning of a cDNA encoding this enzyme. Recombinant NMNAT exhibited catalytic properties similar to the originally purified enzyme. Human NMNAT (molecular weight 31932) consists of 279 amino acids and exhibits substantial structural differences to the enzymes from lower organisms. A putative nuclear localization signal was confirmed by immunofluorescence studies. NMNAT strongly inhibited recombinant human poly(ADP-ribose) polymerase 1, however, NMNAT was not modified by poly(ADP-ribose). NMNAT appears to be a substrate of nuclear kinases and contains at least three potential phosphorylation sites. Endogenous and recombinant NMNAT were phosphorylated in nuclear extracts in the presence of [gamma-(32)P]ATP. We propose that NMNAT's activity or interaction with nuclear proteins are likely to be modulated by phosphorylation.

Obstetrical brachial plexus injuries: incidence, natural course and shoulder contracture.

The incidence of obstetric brachial plexus injury (OBPI) was investigated and the natural course of this disorder and the frequency of shoulder contracture described. Between 1988 and 1997 13,366 children with a gestational age of 30 weeks or more, were born at the Academic Medical Center, Amsterdam. Of these, 62 had an OBPI (4.6 per 1000). Complete neurological recovery occurred in 72.6% of cases. Half of them had a delayed recovery of more than three weeks (mean recovery time 6.2 +/- 3.1 months). Shoulder contracture occurred in at least one-third of the children with delayed recovery and in at least two-thirds of the children with incomplete recovery. The incidence of OBPI in our hospital was found to be higher and to have a less favourable natural course than is usually reported in the literature. Contracture of the shoulder joint is frequently found even in infants with complete neurological recovery.

ATP for the DNA ligation step in base excision repair is generated from poly(ADP-ribose).

In mammalian cells, the base excision repair (BER) pathway is the main route to counteract the mutagenic effects of DNA lesions. DNA nicks induce, among others, DNA polymerase activities and the synthesis of poly(ADP-ribose). It is shown here that poly(ADP-ribose) serves as an energy source for the final and rate-limiting step of BER, DNA ligation. This conclusion was drawn from experiments in which the fate of [(32)P]poly(ADP-ribose) or [(32)P]NAD added to HeLa nuclear extracts was systematically followed. ATP was synthesized from poly(ADP-ribose) in a pathway that strictly depended on nick-induced DNA synthesis. NAD was used for the synthesis of poly(ADP-ribose), which, in turn, was converted to ATP by pyrophosphorylytic cleavage utilizing the pyrophosphate generated from dNTPs during DNA synthesis. The adenylyl moiety was then preferentially used to adenylate DNA ligase III, from which it was transferred to the 5'-phosphoryl end of the nicked DNA. Finally, ligation to the 3'-OH end resulted in the release of AMP. When using NAD, but not poly(ADP-ribose), in the presence of 3-aminobenzamide, the entire process was blocked, confirming poly(ADP-ribosyl)ation to be the essential initial step. Thus, poly(ADP-ribose) polymerase-1, DNA polymerase beta, and ligase III interact with x-ray repair cross-complementing protein-1 within the BER complex, which ensures that ATP is generated and specifically used for DNA ligation.

Obstetric brachial plexus injury: risk factors related to recovery.

OBJECTIVE: To investigate if multivariate risk calculation can discriminate those infants who do not recover after an obstetric brachial plexus injury (OBPI). STUDY DESIGN: All liveborn infants without lethal congenital abnormalities from 1988 through 1996 with a gestational age > or =30 weeks were included. Outcome variables were all OBPI and non-recovered OBPI. Risk calculation was performed by univariate analysis for all infants and by multivariate logistic analysis for all singleton infants delivered vaginally in cephalic presentation. RESULTS: A total of 62 of 13 366 liveborn infants sustained an OBPI (0.46%). Seventeen (27%) did not recover completely. Birth weight, female sex, second stage >60 min, diabetes, multiparity, maternal age and non-Caucasian origin were important risk factors for non-recovered OBPI. A model without birth weight, which can not be measured accurately antepartum, is considerably less effective. Risk factors for all OBPI and for non-recovered OBPI were similar. CONCLUSION: A predictive multivariate model is of limited value due to the low incidence of non-recovered OBPI. However, it may be useful to discriminate individual cases with exceptional risk.

Association of some potential hormone response elements in human genes with the Alu family repeats.

Short interspersed repeats of the Alu family located in promoters of some human genes contain high-affinity binding sites for thyroid hormone receptor, retinoic acid receptor and estrogen receptor. The standard binding sites for the receptors represent variants of duplicated AGGTCA motif with different spacing and orientation (direct, DR, or inverted, IR), and Alu sequences were found to have functional DR-4, DR-2 or variant IR-3/IR-17 elements. In this study we analyzed distribution and abundance of the elements in a set of human genomic sequences from GenBank and their association with Alu repeats. Our results indicate that a major fraction of potentially active DR-4, DR-2 and variant IR-3/IR-17 elements in the genes is located within Alu repeats. Alu-associated DR-2 elements are conserved in primate evolution. However, very few Alu have potential DR-3 glucocorticoid-response elements. Gel-shift experiments with the probe (AUB) corresponding to the consensus Alu sequence just upstream of the RNA polymerase III promoter B-box and containing duplicated AGGTCA motif indicate that the probe interacts in a sequence-specific manner with human nuclear proteins which bind to standard IR-0, DR-1, DR-4 or DR-5 elements. The AUB sequence was also able to promote thyroid hormone-dependent trans-activation of a reporter gene. The results support the view that Alu retroposons played an important role in evolution of regulation of the primate gene expression by nuclear hormone receptors.

A novel human DNA-binding protein with sequence similarity to a subfamily of redox proteins which is able to repress RNA-polymerase-III-driven transcription of the Alu-family retroposons in vitro.

In this study we identified a novel protein which may contribute to the transcriptional inactivity of Alu retroposons in vivo. A human cDNA clone encoding this protein (ACR1) was isolated from a human expression library using South-western screening with an Alu subfragment, implicated in the regulation of Alu in vitro transcription and interacting with a HeLa nuclear protein down-regulated in adenovirus-infected cells. Bacterially expressed ACR1 is demonstrated to inhibit RNA polymerase III (Pol III)-dependent Alu transcription in vitro but showed no repression of transcription of a tRNA gene or of a reporter gene under control of a Pol II promoter. ACR1 mRNA is also found to be down-regulated in adenovirus-infected HeLa cells, consistent with a possible repressor function of the protein in vivo. ACR1 is mainly (but not exclusively) located in cytoplasm and appears to be a member of a weakly characterized redox protein family having a central, highly conserved sequence motif, PGAFTPXCXXXXLP. One member of the family identified earlier as peroxisomal membrane protein (PMP)20 is known to interact in a sequence-specific manner with a yeast homolog of mammalian cyclosporin-A-binding protein cyclophilin, and mammalian cyclophilin A (an abundant ubiquitously expressed protein) is known to interact with human transcriptional repressor YY1, which is a major sequence-specific Alu-binding protein in human cells. It appears, therefore, that transcriptional silencing of Alu in vivo is a result of complex interactions of many proteins which bind to its Pol III promoter.

Stimulation of the catalytic activity of poly(ADP-ribosyl) transferase by transcription factor Yin Yang 1.

The transcriptional regulator Yin Yang 1 (YY1) has previously been demonstrated to physically interact with poly(ADP-ribosyl) transferase (ADPRT). This nuclear enzyme catalyzes the synthesis of ADP-ribose polymers and their attachment to target proteins. It is reported here that YY1 associates preferably with the extensively auto(ADP-ribosyl)ated form of ADPRT, but not with deproteinized ADP-ribose polymers. In the presence of YY1 the catalytic rate of ADPRT is enhanced about 10-fold. This stimulation is in part due to modification of YY1, thus serving as a substrate of the reaction. In addition, automodification of ADPRT is also substantially increased. The activation by YY1 is most pronounced at low concentrations of ADPRT suggesting that the presence of YY1 may either facilitate the formation of catalytically active dimers of ADPRT or lead to the occurrence of active heterooligomers. The potential significance of these observations was verified by analyzing the activity of ADPRT in HeLa nuclear extracts. The endogenous enzyme exhibited an about 10-fold higher activity as compared to the isolated recombinant protein. It is likely that the heat-stable transcription factor YY1 contributed to the increased activity of ADPRT detected in the nuclear extracts, because heated extracts had a similar stimulatory effect on isolated ADPRT as isolated YY1 used at comparable concentrations. It is concluded that YY1 may be an important regulator of ADPRT and, therefore, could support the function of ADPRT to facilitate DNA repair.

Regulation of RNA polymerase II-dependent transcription by poly(ADP-ribosyl)ation of transcription factors.

Poly(ADP-ribosyl) transferase (ADPRT) is a nuclear protein that modifies proteins by forming and attaching to them poly(ADP-ribose) chains. Poly(ADP-ribosyl)ation represents an event of major importance in perturbed cell nuclei and participates in the regulation of fundamental processes including DNA repair and transcription. Although ADPRT serves as a positive cofactor of transcription, initiation of its catalytic activity may cause repression of RNA polymerase II-dependent transcription. It is demonstrated here that ADPRT-dependent silencing of transcription involves ADP-ribosylation of the TATA-binding protein. This modification occurs only if poly(ADP-ribosyl)ation is initiated before TATA-binding protein has bound to DNA and thereby prevents formation of active transcription complexes. Specific DNA binding of other transcription factors including Yin Yang 1, p53, NFkappaB, Sp1, and CREB but not c-Jun or AP-2 is similarly affected. After assembly of transcription complexes initiation of poly(ADP-ribosyl)ation does not influence DNA binding of transcription factors. Accordingly, if bound to DNA, transcription factors are inaccessible to poly(ADP-ribosyl)ation. Thus, poly(ADP-ribosyl)ation prevents binding of transcription factors to DNA, whereas binding to DNA prevents their modification. Considering its ability to detect DNA strand breaks and stimulate DNA repair, it is proposed that ADPRT serves as a molecular switch between transcription and repair of DNA to avoid expression of damaged genes.

A novel function of poly(ADP-ribosyl)ation: silencing of RNA polymerase II-dependent transcription.

Poly(ADP-ribosyl) transferase (ADPRT) is a nuclear enzyme that catalyzes the synthesis of ADP-ribose polymers from NAD+ as well as the transfer of these polymers onto acceptor proteins. The predominant acceptor of the poly(ADP-ribose) chains appears to be the enzyme itself. The function of ADPRT is thought to be related to a number of nuclear processes, including DNA repair and transcription. In this study, it was found that polymerase II-dependent transcription in nuclear HeLa extracts was repressed in the presence of NAD+ at concentrations as low as 1 microM. This repression was strictly dependent on the activity of ADPRT and correlated with the auto(ADP-ribosyl)ation of the enzyme. Subsequent degradation of the ADP-ribose polymers by enzymatic activities present in the nuclear extracts restored transcriptional activity. It would appear from these results that poly(ADP-ribosyl)ation represents the key event of the mechanism underlying NAD(+)-dependent silencing of transcription. Importantly, ADPRT- and NAD(+)-dependent silencing was observed only if poly(ADP-ribosyl)ation had taken place before formation of the transcription complex was completed. That is, if the nuclear extract was preincubated for more than 15 min in the presence of template DNA, transcription was rendered entirely insensitive to NAD+. These results suggest that poly(ADP-ribosyl)ation may prevent polymerase II-dependent transcription, but does not interfere with ongoing transcription. Taking into account the known function of ADPRT, this enzyme may facilitate recovery from DNA damage by stimulating DNA repair and silencing transcription.

Interaction of the transcription factor YY1 with human poly(ADP-ribosyl) transferase.

Poly(ADP-ribosyl) transferase (ADPRT) is a nuclear enzyme that catalyzes the synthesis of ADP-ribose polymers from NAD+ as well as the transfer of these polymers onto acceptor proteins. The function of ADPRT is thought to be related to a number of nuclear processes including DNA repair and transcription. The transcription factor Yin Yang 1 (YY1) is a potent regulator of RNA polymerase II (Pol II)-dependent transcription. In this study Alu-retroposon-associated binding sites for YY1 located in the distal region of the promoter of the human ADPRT gene have been identified suggesting a possible involvement of this protein in the regulation of ADPRT-gene expression. In the presence of the recombinant automodification domain of the ADPRT the formation of specific YY1 complexes, detected in gel-shift experiments, was strongly inhibited, indicating that this domain of the enzyme may interact directly with YY1. In accordance with this result YY1 was specifically precipitated from nuclear extracts by ADPRT immobilized on sepharose. These results suggest a direct ADPRT-YY1 interaction which may be of importance in the regulation of Pol II-dependent transcription. They also indicate that in some human promoters this regulation may be mediated by retroposons of the Alu family.

Protein-protein interaction of the human poly(ADP-ribosyl)transferase depends on the functional state of the enzyme.

Poly(ADP-ribosyl)transferase (pADPRT) is a nuclear protein which catalyzes the polymerization of ADP-ribose using NAD+ as substrate, as well as the transfer of ADP-ribose polymers to itself and other protein acceptors. The catalytic activity of pADPRT strictly depends on the presence of DNA single-strand breaks. In this report, protein-protein interaction of pADPRT was found to depend on both the extent of automodification with poly(ADP-ribose) and the presence of DNA. Specific binding of radiolabeled pADPRT to transblotted proteins was first tested in blot overlay experiments. For radiolabeling, use was made of the ability of the enzyme to incorporate [32P]ADP-ribose from [32P]NAD+. Varying the concentration of NAD+, two different forms of automodified pADPRT were obtained: oligo(ADP-ribosyl)ated pADPRT with less than 20 ADP-ribose units per chain, and poly(ADP-ribosyl)ated pADPRT with polymer lengths of up to 200 ADP-ribose residues. Interaction of these probes with transblotted HeLa nuclear extracts, purified histones, and distinct regions of recombinant pADPRT was investigated. While the oligo(ADP-ribosyl)ated enzyme associated preferentially with transblotted purified histones, or pADPRT present in HeLa nuclear extracts, poly(ADP-ribosyl)ated pADPRT bound to a variety of transblotted proteins in the nuclear extracts. In the presence of DNA, both the oligo- and the poly(ADP-ribosyl)ated enzymes bound to the transblotted recombinant zinc finger domain of pADPRT even at high salt concentrations. In the absence of DNA, the transblotted automodification domain of pADPRT appeared to be the region involved in self-association. In another set of experiments, unmodified or poly(ADP-ribosyl)ated pADPRT was immobilized on Sepharose. Affinity precipitation of recombinant pADPRT domains confirmed the specific interaction of pADPRT with its zinc finger region and the automodification domain, whereas no interaction was observed with the NAD+ binding domain. Affinity precipitation of HeLa nuclear extracts with poly(ADP-ribosyl)ated pADPRT-Sepharose led to the enrichment of a number of proteins, whereas nuclear proteins bound to the unmodified pADPRT-Sepharose in a smaller extent. The results suggest that protein-protein interaction of the human pADPRT is governed by its functional state.

Binding of antibodies against high and low molecular weight cytokeratin proteins in the human placenta with special reference to infarcts, proliferation and differentiation processes.

Recent immunocytochemical studies have shown that placental villous trophoblasts contain the high molecular weight cytokeratin (CK) proteins 5/6 and 17. In the case of CK 17, trophoblastic immunostaining was positive in villi covered by fibrinoid. CKs 5/6 and 17 are expressed by hyperproliferative cells. The aim of this investigation was to examine the location of these CKs in placental infarcts, known to be demarcated by fibrinoid and hyperproliferative trophoblasts. The results were compared with those obtained by immunostaining against Ki-67, tenascin and alpha 1-, alpha 6- and beta 1-integrins, which are involved in cell proliferation, differentiation and regenerative processes. Furthermore, the expression of the single CKs 7, 8, 10, 13, 14, 18 and 19 was investigated by immunocytochemistry and immunoblotting. While low and high molecular weight CKs were present in villous and extravillous trophoblasts, only low molecular weight CKs were detected in vascular and extravascular placental smooth muscle cells. Placental infarcts revealed different immunoreactivities in the infarct margin and centre: high molecular CKs, tenascin, Ki-67 and oncofoetal fibronectin predominated in the infarct margin, low molecular CKs, fibrin and integrins in the centre. The expression of tenascin and a defined change in the expression of CK 17 indicates villous repair and hyperproliferative mechanisms in placental infarcts.