Poly(ADP-ribosyl)ation is catalyzed by NAD + :protein(ADP-ribosyl)transferase (ADPRT), a chromatinassociated enzyme which, in the presence of DNA breaks, transfers ADP-ribose from NAD + to nuclear proteins. This post-translational modification has been implicated in many fundamental processes, like DNA repair, chromatin stability, cell proliferation, and cell death. To elucidate the biological function of ADPRT and poly(ADP-ribosyl)ation in vivo the gene was inactivated in the mouse germ line. Mice homozygous for the ADPRT mutation are healthy and fertile. Analysis of mutant tissues and fibroblasts isolated from mutant fetuses revealed the absence of ADPRT enzymatic activity and poly(ADP-ribose), implying that no poly(ADP-ribosyl)ated proteins are present. Mutant embryonic fibroblasts were able to efficiently repair DNA damaged by UV and alkylating agents. However, proliferation of mutant primary fibroblasts as well as thymocytes following y-radiation in vivo was impaired. Moreover, mutant mice are susceptible to the spontaneous development of skin disease as -30% of older mice develop epidermal hyperplasia. The generation of viable ADPRT-/-mice negates an essential role for this enzyme in normal chromatin function, but the impaired proliferation and the onset of skin lesions in older mice suggest a function for ADPRT in response to environmental stress.

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 31 932) 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 [Q Q-32 P]ATP. We propose that NMNAT's activity or interaction with nuclear proteins are likely to be modulated by phosphorylation. ß 2001 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.

Mitochondrial ADP-ribosylation leads to modi®cation of two proteins of~26 and 53 kDa. The nature of these proteins and, hence, the physiological consequences of their modi®cation have remained unknown. Here, a 55 kDa protein, glutamate dehydrogenase (GDH), was established as a speci®c acceptor for enzymatic, cysteine-speci®c ADP-ribosylation in mitochondria. The modi®ed protein was isolated from the mitochondrial preparation and identi®ed as GDH by N-terminal sequencing and mass spectrometric analyses of tryptic digests. Incubation of human hepatoma cells with [ 14 C]adenine demonstrated the occurrence of the modi®cation in vivo. Puri®ed GDH was ADPribosylated in a cysteine residue in the presence of the mitochondrial activity that transferred the ADP-ribose from NAD + onto the acceptor site. ADPribosylation of GDH led to substantial inhibition of its catalytic activity. The stoichiometry between incorporated ADP-ribose and GDH subunits suggests that modi®cation of one subunit per catalytically active homohexamer causes the inactivation of the enzyme. Isolated, ADP-ribosylated GDH was reactivated by an Mg 2+ -dependent mitochondrial ADP-ribosylcysteine hydrolase. GDH, a highly regulated enzyme, is the ®rst mitochondrial protein identi®ed whose activity may be modulated by ADP-ribosylation. Keywords: ADP-ribosylation/glutamate dehydrogenase/ mitochondria/protein modi®cation IntroductionBesides its role in energy transduction, NAD + serves an important function in the regulation of multiple cellular processes (Ziegler, 2000). This nucleotide may be utilized for post-translational protein modi®cation known as poly- (Oei et al., 1997) or monoADP-ribosylation (Okazaki and Moss, 1996). It is also used as a substrate by NAD + glycohydrolases, which may form cyclic ADP-ribose, a potent intracellular calcium-mobilizing agent (Lee, 1997;Ziegler et al., 1997;Galione et al., 1998). NAD + glycohydrolases also generate free ADP-ribose from either NAD + or cyclic ADP-ribose. Free ADP-ribose, in turn, can react non-enzymatically with protein lysine (CervantesLaurean et al., 1993;Jacobson et al., 1994) or cysteine (McDonald et al., 1992) residues, leading to protein glycation. However, the in vitro systems described so far suggest that this kind of protein modi®cation occurs only at high concentrations of ADP-ribose (Frei and Richter, 1988;McDonald and Moss, 1993a).In vivo, monoADP-ribosylation is catalyzed by monoADP-ribosyl transferases which have been found in eukaryotes and prokaryotes (Moss and Vaughan, 1988). Eukaryotic monoADP-ribosyl transferases modify speci®c amino acids of the acceptor proteins, including arginine (Moss et al., 1980;Zolkiewska et al., 1992), cysteine (Tanuma et al., 1987;Saxty and van Heyningen, 1995;Jorcke et al., 1998) and diphthamide (Lee and Iglewski, 1984) residues. Endogenous ADP-ribosylation also appears to occur in hydroxyl-containing amino acid residues (Cervantes-Laurean et al., 1995). Clostridial C3-like exoenzymes modify Rho proteins at an asparagine residue (Aktories, 1997). Several speci...

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DNAs of adenovirus type 2 and type 12 contain low amounts of methylated bases (0.01 and 0.02% N6-methyladenine per adenine, if any, and 0.04 and 0.06% 5-methylcytosine per cytosine for type 2 and type 12, respectively), whereas the DNA of the mammalian host cells contains much more 5-methylcytosine (3.57% for human KB cells). The Methylation of DNA was determined by a sensitive method based on two consecutive steps of two-dimensional thin-layer chromatography of the radioactively labeled DNA bases. By this procedure the detection limits of 5-methylcytosine and N6-methyladenine could be lowered to 0.01% per main base. During replication of adenoviruses in permissive human cells, a large amount of viral DNA is synthesized in the nucleus of the host cell (1-3). Viral DNA accounts for 30-50% of the total intracellular DNA at late times after infection (2, 3). Concomitantly with the onset of viral DNA replication, host DNA synthesis is drastically reduced (4).In previous DNA base analyses of mammalian cells 3-6% 5-methylcytosine (MeCyt) per cytosine (Cyt) was found (5, 6), while N6-methyladenine (MeAde) could not be detected (7,8).Little is known about the biological role of DNA methylation. There are reports on tissue specificities in the content of MeCyt in mammalian cells (5,6,9). Certain developmental stages are associated with variations in the content of methylated DNA bases in pro-and eukaryotes (10, 11). The only established function of certain DNA methyltransferases is the modification of DNA in bacteria leading to resistance against restriction endonucleases of the same specificity (12, 13).Since it is presumed that synthesis of adenovirus DNA in human cells is mediated, at least in part, by the host replication system, one would expect viral DNA to show the methylation pattern characteristic of host DNA. However, in contrast to host -DNA, adenovirus DNA has a low level of methylation. Comparison of untransformed cells and cells transformed by adenovirus has shown a difference in the levels of DNA methyl- (Calbiochem). The method of further purification has been described (21).In some experiments the total cellular DNA and the nonencapsidated viral DNA were isolated from Ad2-infected KB cells 40-48 hr after infection. Cell extracts were prepared by ultrasonic treatment, and the virions were isolated by equilibrium sedimentation in CsCl density gradients with a mean density of 1.334 g/cm3 as described (3). All free intracellular DNA was recovered from the pellet of this gradient and recentrifuged to equilibrium in a CsCl density gradient with a mean density of 1.70 g/cm3.Purification of Viral and Cellular DNA. The DNA was precipitated with two volumes of ethanol and kept for 4 hr at -20°. Traces of RNA were removed by hydrolysis in 0.25 M KOH 16-18 hr at 37°. Subsequently, the DNA was again pre-3923

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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.

After bacteriophage T7 infection, a protein kinase (EC 2.7.1.37; ATP:protein phosphotransferase) activity can be demonstrated in E. coli in vivo by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Cell-free extracts catalyzed the transfer of the terminal phosphoryl group of [-y_32P]ATP to endogenous protein acceptor or to added histone. The bond between phosphate and protein shows the characteristies of serine phosphate: it is stable in 1 N HCl (1000) and cleaved by 1 N KOH (370) and by alkaline phosphatase treatment. Moreover, after partial acid hydrolysis, radiophosphate migrates with marker O-phosphoserine on polyethyleneimine-cellulose thin-layer chromatograms. Enzyme activity in uninfected cells is negligible. Ultraviolet irradiation of the phage genome prevents the appearance of the protein kinase; irradiation of the host genome does not. The enzyme activity occurs 4 min after infection and its gene maps in the early region (promoter proximal to gene 1). Ribosomal proteins are phosphorylated in vivo and are substrates in vitro. Enzyme activity in vitro is not changed by addition of cyclic AMP or cyclic GMP.Bacteriophage systems have been used extensively to study the regulation of gene expression (1,2). One particular aspect of these systems is the rapidity of changes in the pattern of gene expression; phage T7 induces a whole series of control mechanisms within 4-5 min (2, 3). This rapidity of change in gene expression prompted our search for group transfers after phage infection (4-6). A phage-induced group transfer to components of the existing host machinery of gene expression would allow both a fast and an economical control.In our previous work on phage T7, we reported that labeled phosphate is incorporated into ribosomal protein after T7 infection (6,23 were then subject to SDS slab-gel electrophoresis (9); the gels were dried and placed on x-ray film overnight. Assay for Protein Kinase In Vitro. Brij-lysozyme cell extracts (10) were used as enzyme sources. The incubations were carried out at 300 for 5 min in a total volume of 0.1 ml. The incubation mixtures contained 30A]. of cell extract, 3 mg/ml of type II histone, and either (1) 15 mM sodium phosphate (pH 7.0), 0.01 MMgCl2, 0.14 mM ATP (35.7 Ci ['y32-P]ATP/mol) or (2) 25 mM Tris HCl (pH 7.0), 5 mM MgCI2, 0.1 mM EDTA, 44,AM, ATP (114 Ci of [32P]ATP/mol). The incubations were started by the addition of enzyme (cell extract) and stopped by addition of 1.5 ml of 10% trichloroacetic acid.The precipitates were boiled in trichloroacetic acid for 15 min, chilled in ice for 10 min, and collected on glass-fiber filter discs. The discs were washed 5 times with 10 ml of 10% trichloroacetic acid and once with 10 ml of methanol. After the filters were dried, radioactivity on the filters was measured by liquid scintillation spectrometry. In selected experiments, the 10% trichloroacetic acid suspensions of precipitate were heated to 900 for 20 min, cooled to ambient temperature, and centrifuged at 2500 X g. These precipit...