Functional Characterization of the Poly(ADP-ribose) Polymerase Activity of Tankyrase 1, a Potential Regulator of Telomere Length
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Poly(ADP-ribose) polymerase (PARP) polyADP-ribosylates proteins involved in various physiological processes. Accumulated evidence suggests not only protein-conjugated poly(ADP-ribose) but also protein-free poly(ADP-ribose) function in various physiological processes. There are increasing occasions that require protein-free poly(ADP-ribose) to study the function and dynamics of poly(ADP-ribose) in cells. However, the availability of poly(ADP-ribose) is still limited because a chemical synthesis method has not been established. Here, we describe an improved method for the preparation of protein-free poly(ADP-ribose), synthesized enzymatically by using a recombinant PARP-1 expression system and purified with an anion-exchange column chromatography. This method will be useful for biochemical and biological investigation of poly(ADP-ribose) functions and dynamics.
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Abstract We have characterized the biochemical association of two DNA damage‐dependent enzymes, poly(ADP‐ribose) polymerase‐1 (PARP‐1) [EC 2.4.2.30] and DNA polymerase β (pol β ) [2.7.7.7]. We reproducibly observed that pol β is an efficient covalent target for ADP‐ribose polymers under standard conditions of enzymatically catalyzed ADP‐ribosylation of β NAD + as a substrate. The efficiency of poly(ADP‐ribosyl)ation increased as a function of the pol β and β NAD + concentrations. To further characterize the molecular interactions between these two unique polymerases, we also subjected human recombinant PARP‐1 to peptide‐specific enzymatic degradation with either caspase‐3 or caspase‐7 in vitro. This proteolytic treatment, commonly referred to as ‘a hallmark of apoptosis’, generated the two physiologically relevant peptide fragments of PARP‐1, e.g. , a 24‐kDa amino‐terminus and an 89‐kDa carboxy‐terminal domain. Interestingly, co‐incubation of the two peptide fragments of PARP‐1 with full‐length pol β resulted in their domain‐specific molecular association as determined by co‐immunoprecipitation and reciprocal immunoblotting. Therefore, our data strongly suggest that, once PARP‐1 is proteolyzed by either caspase‐3 or caspase‐7 during cell death, the specific association of its apoptotic fragments with DNA repair enzymes, such as pol β , may serve a regulatory molecular role in the execution phase of apoptosis.
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Poly(ADP-ribose) polymerase-1 consumes NAD+ to catalyze poly(ADP-ribosyl)ation of target proteins, which modulates various biological functions. However, excessive poly(ADP-ribose) polymerase-1 activation mediates oxidative cell death. Our recent studies have indicated that NAD+ can enter into astrocytes to prevent poly(ADP-ribose) polymerase-1 cytotoxicity. In this study, we show that NADH can also enter into astrocytes, which can significantly decrease poly(ADP-ribose) polymerase-1-induced astrocyte death even when applied 3–4 h after poly(ADP-ribose) polymerase-1 activation. The protective effects can be produced by 10 μM NADH, which is significantly lower than that required for NAD+ to be protective. These results provide novel information suggesting that NADH can be used for decreasing poly(ADP-ribose) polymerase-1 toxicity, and extracellular NADH can enter into astrocytes to influence cellular functions.
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Single-strand breaks are the commonest lesions arising in cells, and defects in their repair are implicated in neurodegenerative disease. One of the earliest events during single-strand break repair (SSBR) is the rapid synthesis of poly(ADP-ribose) (PAR) by poly(ADP-ribose) polymerase (PARP), followed by its rapid degradation by poly(ADP-ribose) glycohydrolase (PARG). While the synthesis of poly(ADP-ribose) is important for rapid rates of chromosomal SSBR, the relative importance of poly(ADP-ribose) polymerase 1 (PARP-1) and PARP-2 and of the subsequent degradation of PAR by PARG is unclear. Here we have quantified SSBR rates in human A549 cells depleted of PARP-1, PARP-2, and PARG, both separately and in combination. We report that whereas PARP-1 is critical for rapid global rates of SSBR in human A549 cells, depletion of PARP-2 has only a minor impact, even in the presence of depleted levels of PARP-1. Moreover, we identify PARG as a novel and critical component of SSBR that accelerates this process in concert with PARP-1.
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Metabolic stability
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Poly( ADP-ribose) polymerase 1 (PARP-1) plays an essential role in DNA repair and maintenance of homeostasis and genome stability. Increased PARP-1 activity has been reported in various forms of cancer. Thus the absence of PARP-1 or the use of its inhibitors depresses DNA repair function, sensitizes cancer cells to DNA damage and enhances the therapeutic effect of radio-or chemotherapy. PARP-1 is potentially a therapeutic target of malignant tumors.
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Poly(ADP-ribose) polymerases; DNA repair enzymes; Neoplasms; Poly (ADP-ribose) polymerases inhibitors
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