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M E Smulson

Publications and source records attributed to M E Smulson.

At least 55 records · Page 3Linked to original sources
PubMed1990

Cell cycle regulation of an exogenous human poly(ADP-ribose) polymerase cDNA introduced into murine cells.

We have evaluated the regulation of expression of the poly(ADP-ribose) polymerase gene during cell growth and replication. In a synchronized population of HeLa cells or in serum-stimulated WI-38 cells, steady-state levels of the polymerase mRNA were highest at late S and S-G2 phases and negligible in early S phase. Transcription did not solely account for the significant increase in the mRNA levels observed in late S phase by Northern analysis. The stability of the mRNA was dependent upon the percent proliferating cells in the culture. Accordingly, polymerase mRNA from cells in early exponential phase was significantly more stable than from cells in stationary phase of asynchronous growth. To clarify these observations, we utilized a novel heterologous expression system that involved murine 3T3 cells transfected with a human poly(ADP-ribose) polymerase cDNA under the control of a non-cell cycle-specific promoter. Cells were synchronized, and a comparison was made of the endogenous (murine) and exogenous (human) polymerase mRNA levels. Both the endogenous and the exogenous mRNA were specifically stabilized by the same mechanisms and only during late S phase; therefore, we concluded that mRNA pools for the polymerase are regulated at the post-transcriptional level. The heterologous expression system confirmed that the post-transcriptional regulation system in the mouse cells can recognize and faithfully regulate the human cDNA in response to the murine cell cycle signals. More importantly, the presence of extra copies (human) of the polymerase gene did not provide an increased amount of the total polymerase mRNA or protein and, in fact, the sum of the endogenous and exogenous mRNA in the transfected cells was approximately the same as the level of endogenous transcript in the control cells. This suggested that there might be a limit to the amount of polymerase protein accumulating in the cellular pool and thus levels of poly(ADP-ribose) polymerase may be autoregulated.

Animals↗
PubMed1990

Expression of the poly(ADP-ribose) polymerase gene following natural and induced DNA strand breakage and effect of hyperexpression on DNA repair.

The catalytic activity of the nuclear enzyme poly(ADP-ribose) polymerase (NAD+ ADP-ribosyl transferase, EC 2,4,2,30) is totally dependent upon the presence of DNA strand breaks. Having isolated a full-length cDNA for the polymerase, we have now evaluated the effect of endogenously and exogenously induced DNA strand breaks on the transcriptional control of this enzyme. During retinoic acid or dimethyl-sulfoxide-induced differentiation of HL-60 human leukemia cells, which may involve DNA breaks as well as other changes in chromatin, mRNA levels for the polymerase increased very early and remained high for up to 48 h after which it decreased to pre-induced levels. Polymerase transcript levels did not change, however, during the induction of DNA strand breaks by dimethylsulfate, a variety of other alkylating agents, X-irradiation, or UV-irradiation in several mammalian cell lines. It appears that in sharp contrast to the catalytic requirement of the polymerase, the induction of transcription of the polymerase gene may not be a strand-break-dependent process. The noninducibility of the polymerase gene following DNA damage suggested that there may be adequate levels of the polymerase in the cells to cope with DNA damage. To test this hypothesis we examined the efficacy of DNA repair in Cos cells engineered to overexpress the polymerase. Although there was a slight augmentation of the repair rate, this increase was apparent only after very high levels of DNA damage and only at early repair times. After a longer repair period, the extent of repair in control cell was similar to that in the cell overexpressing the polymerase. We thus conclude that the basal levels of the polymerase are adequate for significant amounts of DNA damage.

Animals↗
PubMed1987

In vivo characterization of the poly(ADP-ribosylation) of SV40 chromatin and large T antigen by immunofractionation.

We have confirmed the poly(ADP-ribosylation) of large T antigen of SV40 by using antibodies to both large T antigen and poly(ADP-ribose) and consequently have begun to characterize how this post-translational nuclear modification of the viral protein modulates its biological functions. SV40 minichromosomal subpopulation containing replicative intermediate DNA was shown to have a significantly higher affinity for anti-poly(ADP-Rib)-Sepharose than viral chromatin fractions containing mature minichromosomal DNA. An anti-large T-Sepharose column was used to isolate T antigen from crude extracts by two different approaches: (1) large T antigen was labeled with [35S]methionine in vivo and the infected cell extract was immunofractionated to isolate large T antigen and (2) large T antigen from infected cell extracts was immunofractionated followed by immunostaining. Using these techniques, 1-10% of the total T antigen from infected cells was found to be poly(ADP-ribosylated). Minichromosome preparations per se were also subjected to immunofractionation on anti-large T-Sepharose. The high level of retention of poly(ADP-ribosylated) species of minichromosomes on this matrix suggested that this post-translational modification of viral chromatin may be related to those steps in viral replication and transcription under regulation by large T antigen.

Animals↗
PubMed1987

cDNA sequence, protein structure, and chromosomal location of the human gene for poly(ADP-ribose) polymerase.

Recently we described a full-length cDNA for the human nuclear enzyme poly(ADP-ribose) polymerase. Here, we report the chromosomal localization and partial map of the human gene for this enzyme as well as the complete coding sequence for this protein. The nucleotide sequence reveals a single 3042-base open reading frame encoding a protein with a predicted Mr of 113,135. A comparison of this deduced amino acid sequence with the amino acid sequence of three peptides derived from human poly(ADP-ribose) polymerase revealed a match of 27 amino acid residues. A computer-derived structural analysis of the enzyme and a search for similarities with other proteins confirmed that the polymerase belongs to a subfamily of DNA/NAD-binding proteins and DNA-repair proteins. Possible Zn2+-binding "fingers," a nucleotide-binding fold, and a nuclear transport signal were noted. Additionally, chromosomal mapping has identified polymerase-hybridizing sequences on human chromosomes 1 (the active gene), 13, and 14 (processed pseudogenes). Using the polymerase cDNA as a probe, we also have detected several DNA restriction fragment length polymorphisms in normal humans.

Amino Acid Sequence↗
PubMed1987

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

cDNAs encoding poly(ADP-ribose) polymerase from a human hepatoma lambda gt11 cDNA library were isolated by immunological screening. One insert of 1.3 kilobases (kb) consistently hybridized on RNA gel blots to an mRNA species of 3.6-3.7 kb, which is consistent with the size of RNA necessary to code for the polymerase protein (116 kDa). This insert was subsequently used in both in vitro hybrid selection and hybrid-arrested translation studies. An mRNA species from HeLa cells of 3.6-3.7 kb was selected that was translated into a 116-kDa protein, which was selectively immunoprecipitated with anti-poly (ADP-ribose) polymerase. To confirm that the 1.3-kb insert from lambda gt11 encodes for poly(ADP-ribose) polymerase, the insert was used to screen a 3- to 4-kb subset of a transformed human fibroblast cDNA library in the Okayama-Berg vector. One of these vectors [pcD-p(ADPR)P; 3.6 kb] was tested in transient transfection experiments in COS cells. This cDNA insert contained the complete coding sequence for polymerase as indicated by the following criteria: A 3-fold increase in in vitro activity was noted in extracts from transfected cells compared to mock or pSV2-CAT transfected cells. A 6-fold increase in polymerase activity in pcD-p(ADPR)P transfected cell extracts compared to controls was observed by "activity gel" analysis on gels of electrophoretically separated proteins at 116 kDa. A 10- to 15-fold increase in newly synthesized polymerase was detected by immunoprecipitation of labeled transfected cell extracts. Using pcD-p(ADPR)P as probe, it was observed that the level of poly(ADP-ribose) polymerase mRNA was elevated at 5 and 7 hr of S phase of the HeLa cell cycle, but was unaltered when artificial DNA strand breaks are introduced in HeLa cells by alkylating agents.

Carcinoma, Hepatocellular↗
PubMed1986

Relationship between DNA strand breaks and inhibition of poly (ADP-ribosylation): enhancement of carcinogen-induced transformation.

Inhibition of poly(ADP-Rib) by benzamide (BA) or 3-amino-benzamide (3AB) for a limited period (i.e., when ADP-ribosylation is elevated) during and shortly following X-ray or MNNG-induced DNA damage of BALB/3T3 cells significantly (3- to 30-fold) enhanced transformation frequency by these agents. Individual Type III foci isolated from benzamide, X-ray, or X-ray plus benzamide treated cultures were established and characterized for growth in soft agar and for tumor induction in nude mice. DNA isolated from representative transformed lines established as a result of BA, X-ray or X-ray and BA treatments was transfected onto NIH/3T3 cells. Transformation efficiencies ranging from 0.17 to 0.28 foci/micrograms of DNA were observed suggesting the possibility that dominant transforming gene(s) were responsible for the oncogenic phenotype of radiation and benzamide transformed DNA.

Animals↗
PubMed1985

Selective isolation of domains of chromatin proximal to both carcinogen-induced DNA damage and poly-adenosine diphosphate-ribosylation.

Poly-adenosine diphosphate (ADP)-ribosylation of nuclear proteins has been demonstrated previously to be activated in vivo by the presence of DNA single-strand breaks and has thus been implicated to play an important role in altering chromatin structure during cellular recovery from DNA damage. Based upon these considerations, a novel immunofractionation method, using antipoly(ADP-ribose) coupled to Sepharose, has been used to enrich for those limited domains of chromatin undergoing poly-ADP-ribosylation. We have used three independent methods to verify the presence of significant levels of single-strand DNA breaks adjacent to polynucleosomes engaged in ADP-ribosylation.

Chromatin↗
PubMed1984

Association of poly(adenosine diphosphate ribosylated) nucleosomes with transcriptionally active and inactive regions of chromatin.

We have investigated whether transcriptionally active or inactive gene sequences are associated in vivo with poly(adenosine diphosphate ribosylated) regions of chromatin. Soluble HeLa cell chromatin derived from nuclei treated either briefly or extensively with micrococcal nuclease was fractionated on an anti-poly(adenosine diphosphate ribose)-Sepharose column [Malik, N., Miwa, M., Sugimura, T., Thraves, P., & Smulson, M. E. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 2554-2558] to obtain fractions that were enriched or depleted in poly(ADP-ribosylated) chromatin. DNA obtained from these fractions was then probed for active and inactive gene sequences with a cDNA probe made from total cell mRNA and a probe for the beta-globin gene. Chromatin enriched in poly(ADP-ribosylated) nucleosomes contained both active and inactive gene sequences as detected by the probes and appeared to be more nuclease sensitive than that found in the fraction of chromatin depleted of poly(ADP-Rib). Poly(ADP-ribosylated) chromatin from nuclei digested briefly with nuclease showed an enrichment in both active and inactive genes while that treated extensively with nuclease showed either no enrichment or a depletion of active and inactive genes. Actively transcribed chromatin was digested at a rate several times that of the bulk or inactive chromatin. Nevertheless, the enrichment of active genes in poly(ADP-ribosylated) nucleosomes derived from brief nuclease digestion was greater than that of inactive genes. These results are interpreted as showing that some, but not all, of actively transcribed chromatin contains associated poly(ADP-ribosylated) proteins. However, since poly(ADP-ribosylated) proteins are also associated with inactive genes, the function of this modification cannot be assigned solely to transcription.

Cell Fractionation↗
PubMed1982

Acceptors for the poly ADP-ribosylation modification of chromatin structure are altered by carcinogen-induced DNA damage.

Poly(ADP-Rib) polymerase is activated by strand breaks in DNA and appears to play an important role in DNA repair. The enzyme catalyses the poly ADP-ribosylation of histones and non-histone proteins, yet the contribution of these major alterations in chromatin composition have, as yet, not been critically evaluated with regard to DNA strand breaks. In the present study, the effects of N-methyl-N-nitrosourea (MNU) upon the poly ADP-ribosylation of nuclear protein acceptors have been identified and quantified at the oligonucleosomal level of chromatin. Treatment of HeLa cells with MNU (4.5 mM) for 1 h resulted in a reduction in the cellular NAD pool (30%), a 2-3 fold stimulation of poly ADP-ribosylation in isolated nuclei and in isolated oligonucleosomes. Of acceptors modified, the automodification of the polymerase was stimulated at least 3-fold. Analysis of the acid-soluble acceptors showed a stimulation in the modification of the core histones and a 2-fold increase in histone H1 poly ADP-ribosylation. This modification causes a novel crosslinking of the latter histone, and this has been studied as it relates to DNA strand breaks in the present work. In vivo treatment with MNU resulted in the synthesis of longer chain or more complex polymer species at the expense of the shorter chained ADP-ribose moieties.

Chromatin↗
PubMed1982

Alterations in the polynucleosomal structure of chromatin by poly ADP-ribosylation of nuclear proteins.

The chromatin-associated enzyme, poly (ADP-Rib) polymerase causes an NAD-dependent aggregation of modified oligonucleosomes as demonstrated by electrophoretic and sedimentation analysis. It has been speculated that poly ADP-ribosylation of histone H1 and its subsequent crosslinking may be crucial in this aggregation. The present work describes some approaches we have taken to investigate this hypothesis. We have characterized the synthesis of poly (ADP-Rib)-H1 dimer in isolated nucleosomes, and using a new immunological method have demonstrated the occurrence of this H1 adduct in living cells. The faithful reassociation of histone H1 with chromatin previously depleted of this histone, has been a major aim of our investigations in order to study the above phenomenon. Our approach has been to monitor this reconstitution by the accuracy of poly ADP-ribosylation of this histone. This criterion was assumed to be a highly sensitive indicator of H1-H1 alignment along oligonucleosome fibers, since a crosslinked dimer of H1 is one product of this reaction. Of special interest was the observation that the removal of H1 from nucleosomes abolished the NAD-promoted aggregation of poly ADP-ribosylated chromatin. This activity could be totally restored with the reconstitution of histone H1. The results suggest a major role for histone H1 and poly ADP-ribosylation in chromatin organization.

Chromatin↗
PubMed1980

Characterization of poly(ADP-ribose)--histone H1 complex formation in purified polynucleosomes and chromatin.

Poly(ADP-ribose) [poly(ADP-Rib)] polymerase of HeLa nucleosomes has been shown in vitro, to catalyze the synthesis of a complex of histone H1 containing 2 H1 histones and 15-16 units of oligo(ADP-Rib). The synthesis of the H1 complex in vitro was compared in polynucleosome populations of various sizes (3--16 and greater than 30) released from HeLa nuclei following micrococcal nuclease digestion. Poly(ADP-Rib) was synthesized from [32P]NAD and the poly(ADP-ribosyl)ation of H1 was studied by selective H1 extraction, gel electrophoresis and autoradiography. Quantitative differences in H1 complex formation occurred when either chromatin concentration or polynucleosome length was varied. The data indicated that H1 complex formation in vitro was favored in polynucleosomes 16 nucleosomes long as compared to 8 nucleosomes. A series of partially ADP-ribosylated H1 species was also detected. Partially modified H1 species migrate more slowly than pure H1 in dodecylsulfate gels. The reduced mobility is a function of the number of attached ADP-Rib moieties. Thus, molecules containing one molecule of H1 and various numbers of ADP-Rib residues can be separated. When the partially modified H1 species were incubated in alkali to cleave the linkage of ADP-Rib to protein, (ADP-Rib1-15) were detected by chain length analysis on 15% polyacrylamide gels. The intermediate H1 species could be chased, in vitro, into as H1 complex with NAD and thus were determined to be successive precursors in the formation of the H1 complex. Evidence is presented that the H1 complex is synthesized in intact cells permeabilized with lysolecithin.

Chromatin↗
PubMed1980

Influence of hydrocortisone on the binding of nitrosoureas to nuclear chromatin subfractions.

The effects of steroid-induced modifications of chromatin structure on the extent and sites of chloroethylnitrosourea binding to chromatin were studied using log-phase HeLa cells. The cells were exposed to 0.1 to 2.0 microM hydrocortisone for 22 hr; this resulted in depressed DNA synthesis while transcriptional activity was stimulated. Hydrocortisone had no effect upon cellular or nuclear uptake of the two nitrosoureas under study, 0.6 mM chlorozotocin or 1-(2-chloroethyl-3-cyclohexyl-1-nitrosourea). Both drugs were found to alkylate transcriptional chromatin preferentially, as demonstrated by DNase II and DNase I digestion. This alkylation was stimulated 2-fold by the same micromolar concentrations of hydrocortisone, 0.1 to 2.0 microM, which stimulated transcription. The extent of nuclear RNA alkylation, determined using RNase T2 as a probe, was found to contribute less than 20% of total chromatin alkylation and was unaffected by steroid pretreatment. Instead, the increased alkylation within these chromatin subfractions was attributed to a steroid-induced alteration of chromatin structure. Electron microscopic examination of HeLa nuclear morphology revealed a hydrocortisone-induced disaggregation of nuclear membrane-associated heterochromatin resulting in a more heterogeneous, less condensed distribution of chromatin. Such data are consistent with a relaxation of the supercoiled chromatin structure, resulting in increased transcription and increased accessibility of potential target sites for nitrosourea alkylation.

Alkylation↗
PubMed1979

Nucleosome periodicity in HeLa cell chromatin as probed by micrococcal nuclease.

When HeLa cell nuclei were treated with micrococcal nuclease (nucleate 3-oligonucleotidohydrolase, EC 3.1.4.7), lysed, and centrifuged, the supernatant from early digests contained two predominant classes of polynucleosomes of repeat size 8N and 16N. With increasing digestion time, the 16 N polynucleosome appeared to be cleaved to the 8N species and finally to the basic subunit of chromatin. The size of the polynucleosomes has been determined by DNA analysis and on polyacrylamide electrophoretic gels of native chromatin particles. The 16N polynucleosome appears to be a unique higher ordered structural component of HeLa cell chromatin. Our recent report, showing that the nuclear protein-modifying enzyme poly(ADP-ribose) polymerase increases in specific activity progressively with increasing nucleosome repeat size up to 8-10N, has been extended in the present study. Activity was also elevated in the polynucleosomes of the 16N structure preferentially cleaved by micrococcal nuclease, although specific activity of the enzyme was highest in octanucleosomes. Acceptors for poly(ADP-ribose) have also been determined in these particles.

Chromatin↗
PubMed1979

Effect of 1-methyl-1-nitrosourea on poly(adenosine diphosphate-ribose) polymerase activity at the nucleosomal level.

The stimulation of poly(adenosine diphosphate ribose) [poly(ADP-ribose)] polymerase activity at the nuclear level after damage of HeLa cells by 1-methyl-1-nitrosourea has been previously reported. We have observed a similar activation of the enzyme after treatment of cells with MNU at the nucleosomal level of chromatin (greater than 1N). This stimulation of enzyme activity did not occur through an inhibition of the glycohydrolase enzyme which cleaves poly(ADP-ribose), or elongation of poly(ADP-ribose) chains, or an increased biosynthesis of enzyme protein. The increased activity appears to be a consequence of the generation of more acceptor sites on nuclear proteins for initiation of poly(ADP-ribose) synthesis. The data indicate that MNU increased the accessibility of nucleosome core histones for modification by poly(ADP) ribosylation.

Chromatin↗