Poly(ADP-ribose) polymerase-1: A Regulator of Protein–Nucleic Acid Interactions in Processes Responding to Genotoxic Impact

Poly(ADP-ribose) polymerase-1 (PARP-1), a nuclear protein of higher eukaryotes, specifically detects strand breaks in DNA. The enzyme is activated in the presence of such breaks and synthesizes poly(ADP-ribose) covalently bound to certain proteins, with PARP-1 itself being the main acceptor. This protein is involved in the majority of DNA-dependent processes, including replication, recombination, repair, and cell death (apoptosis and necrosis). Poly(ADP-ribosyl)ation of proteins is regarded as a mechanism which induces a signal of DNA damage and modulates the function of proteins in response to genotoxic actions. Attention in this review is focused on the role of PARP-1 and poly(ADP-ribosyl)ation in base excision repair (BER), the main process of DNA break repair. The main putative functions of PARP-1 in this process are also considered, namely, its functions as a factor initiating the BER protein complex, a temporary protector of DNA ends, a factor modulating chromatin structure through poly(ADP-ribosyl)ation of histones, and a signal in the mechanism recognizing the degree of DNA damage in the cell.     

Metabolism of NAD+ in Nuclei of Saccharomyces cerevisiae during Stimulation of Its Biosynthesis by Nicotinamide

The activities of nuclear enzymes involved in NAD⁺ metabolism in Saccharomyces cerevisiae strain 913a-1 and its mutant 110 previously selected as an NAD⁺ producer were investigated. The presence of extracellular nicotinamide increased the total NAD⁺ pool in the cells and increased [³H]nicotinic acid incorporation; however, NAD⁺ concentration in isolated nuclei decreased slightly. The stimulating effect of nicotinamide on intracellular synthesis of NAD⁺ correlated with increases in ADP-ribosyl transferase, NAD⁺-pyrophosphorylase, and NAD⁺ ase activities.     

Cyclic ADP-ribose as a potential second messenger for neuronal Ca2+ signaling

Cyclic ADP-ribose (cADPR), a known endogenous modulator of ryanodine receptor Ca2+ releasing channels, is found in the nervous system. Injection of cADPR into neuronal cells primarily induces a transient elevation of intracellular Ca2+ concentration ([Ca2+]i), and/or secondarily potentiates [Ca2+]i increases that are the result of depolarization-induced Ca2+ influx. Acetylcholine release from cholinergic neurons is facilitated by cADPR. cADPR modifies K+ currents or elicits Ca2+-dependent inward currents. cADPR is synthesized by both membrane-bound and cytosolic forms of ADP-ribosyl cyclase in neuronal cells. cADPR hydrolase activity is weak in the membrane fraction, but high in the cytoplasm. Cytosolic ADP-ribosyl cyclase activity is upregulated by nitric oxide/cyclic GMP-dependent phosphorylation. Stimulation of muscarinic and beta-adrenergic receptors activates membrane-bound ADP-ribosyl cyclase via G proteins within membranes of neuronal tumor cells and cortical astrocytes. These findings strongly suggest that cADPR is a second messenger in Ca2+ signaling in the nervous system, although many intriguing issues remain to be addressed before this identity is confirmed.     

Poly(ADP-ribose) Polymerase-1 Inhibits Strand-Displacement Synthesis of DNA Catalyzed by DNA Polymerase β

Poly(ADP-ribose) polymerase-1 (PARP-1), a eucaryotic nuclear DNA-binding protein that is activated by breaks in DNA chains, may be involved in the base excision repair (BER) because DNAs containing single-stranded gaps and breaks are intermediates of BER. The effect of PARP-1 on the DNA synthesis catalyzed in vitro by DNA polymerase beta (pol beta) was studied using analogs of DNA substrates produced during BER and imitating intermediates of the short patch and long patch subpathways of BER. Oligonucleotide duplexes of 34 bp that contained a mononucleotide gap or a single-strand break with tetrahydrofuran phosphate or phosphate at the 5;-end of the downstream oligonucleotide were taken as DNA substrates. The efficiency of DNA synthesis was determined at various ratios of pol beta and PARP-1. The efficiency of gap filling was decreased in the presence of PARP-1, but strand-displacement DNA synthesis was inhibited significantly stronger, which seemed to be due to competition between PARP-1 and pol beta for DNA. In the presence of NAD+ and single-strand breaks in DNA, PARP-1 catalyzes the synthesis of poly(ADP-ribose) covalently attached to the enzyme, and this automodification is thought to provide for dissociation of PARP-1 from DNA. The effect of PARP-1 automodification on inhibition of DNA synthesis was studied, and efficiency of mononucleotide gap filling was shown to be restored, but strand-displacement synthesis did not revert to the level observed in the absence of PARP-1. PARP-1 is suggested to regulate the interaction between pol beta and DNA, in particular, via its own automodification.     

Expression of the soluble adenylyl cyclase during rat spermatogenesis: evidence for cytoplasmic sites of cAMP production in germ cells

To gain insight into the mechanisms of cAMP signaling in germ cells, the expression and subcellular localization of the full-length form of the soluble adenylyl cyclase (sAC) was investigated during rat spermatogenesis and in spermatozoa. A full-length sAC-specific antibody was generated by using a glutathione S-transferase (GST)-sAC carboxyl-terminal region (1399aa-1608aa) fusion protein as the antigen. The selectivity of the purified antibody was confirmed by immunoblotting with lysates from HEK293 cells overexpressing full-length sAC or truncated sAC. Western blot analysis demonstrated that full-length sAC protein appeared on day 25 during testis development. The expression levels increased progressively on days 30 and 35 and remained elevated in adult testis. Full-length sAC protein is retained in spermatozoa from the cauda epididymis. Consistent with the timing of the appearance of the Western blot signal, immunohistochemistry with testis sections at different stages of development detected sAC in late pachytene spermatocytes as well as round and elongating spermatids. Further experiments on the subcellular localization of native or recombinant enzymes revealed that full-length sAC is not only recovered in soluble fractions but also in particulate fractions of testis extracts. Immunofluorescence detection showed localization of the protein in the cytoplasm as well as in organelles of pachytene spermatocytes and spermatids. These findings indicate that cAMP production in spermatids and spermatozoa may occur at sites other than the plasma membrane and suggest that full-length sAC may play a role during spermatid differentiation.     

Specificity of reversible ADP-ribosylation and regulation of cellular processes

Proper and timely regulation of cellular processes is fundamental to the overall health and viability of organisms across all kingdoms of life. Thus, organisms have evolved multiple highly dynamic and complex biochemical signaling cascades in order to adapt and survive diverse challenges. One such method of conferring rapid adaptation is the addition or removal of reversible modifications of different chemical groups onto macromolecules which in turn induce the appropriate downstream outcome. ADP-ribosylation, the addition of ADP-ribose (ADPr) groups, represents one of these highly conserved signaling chemicals. Herein we outline the writers, erasers and readers of ADP-ribosylation and dip into the multitude of cellular processes they have been implicated in. We also review what we currently know on how specificity of activity is ensured for this important modification.     

cGMP对细胞功能的调控

cGMP作为胞内第二信使,主要通过cGMP依赖的蛋白激酶(cGPK),cGMP门控的离子通道,cGMP调节的环核苷酸磷酸二酯酶以及ADP核糖环化酶等后续途径,参与许多细胞功能的调控.cGMP既可以通过蛋白磷酸化,也通过与蛋白磷酸化不直接相关的信号传递途径来调控各类细胞的特定生理功能.     

Poly(ADP-ribosyl)ation-dependent Transient Chromatin Decondensation and Histone Displacement following Laser Microirradiation

Chromatin undergoes a rapid ATP-dependent, ATM and H2AX-independent decondensation when DNA damage is introduced by laser microirradiation. Although the detailed mechanism of this decondensation remains to be determined, the kinetics of decondensation are similar to the kinetics of poly(ADP-ribosyl)ation. We used laser microirradiation to introduce DNA strand breaks into living cells expressing a photoactivatable GFP-tagged histone H2B. We find that poly(ADP-ribosyl)ation mediated primarily by poly(ADP-ribose) polymerase 1 (PARP1) is responsible for the rapid decondensation of chromatin at sites of DNA damage. This decondensation of chromatin correlates temporally with the displacement of histones, which is sensitive to PARP inhibition and is transient in nature. Contrary to the predictions of the histone shuttle hypothesis, we did not find that histone H1 accumulated on poly(ADP-ribose) (PAR) in vivo. Rather, histone H1, and to a lessor extent, histones H2A and H2B were rapidly depleted from the sites of PAR accumulation. However, histone H1 returns to chromatin and the chromatin recondenses. Thus, the PARP-dependent relaxation of chromatin closely correlates with histone displacement.