Functional expression of human poly(ADP‐ribose) polymerase in Schizosaccharomyces pombe results in mitotic delay at G₁, increased mutation rate, and sensitization to radiation

Abstract

The activity of poly(ADP-ribose) polymerase (PADPRP), a chromatin-associated enzyme present in most eukaryotic cells, is stimulated by DNA strand breaks, suggesting a role for the enzyme in the cellular response to DNA damage. However, the primary function of PADPRP remains unknown. We have selected Schizosaccharomyces pombe as a simple eukaryotic system in which to study PADPRP function because this fission yeast shares with mammalian cells important cellular features possibly associated with poly-(ADP-ribos)ylation pathways. We investigated the existence of an endogenous yeast PADPRP by DNA and RNA hybridization to mammalian probes under low-stringency conditions and by PADPRP activity assays. Our data indicate that fission yeasts are naturally devoid of PADPRP. We therefore isolated S. pombe strains expressing PADPRP by transformation with a human full-length PADPRP cDNA under the control of the SV40 early promoter. The human PADPRP construct was transcribed and translated in S. pombe, generating a major transcript of the same size (3.7 kb) as that detected in mammalian cells and a 113-kDa polypeptide, identical in size to the native human PADPRP protein. Yeast recombinant PADPRP was enzymatically active and was recognized by antibodies to human PADPRP. S. pombe cells expressing PADPRP (SPT strains) showed a stable phenotype that was characterized by: (i) cell cycle retardation as a result of a specific delay at the G1 phase, (ii) decreased cell viability in stationary cultures, (iii) enhanced rates of spontaneous and radiation-induced ade6-ade7 mutations, and (iv) increased sensitivity to radiation. SPT strains may prove efficient tools with which to investigate PADPRP functions in eukaryotic cells.

Role of Poly(ADP-ribose) Polymerase (PARP) Cleavage in Apoptosis

“…Osteosarcoma cells expressing mut-PARP exhibited normal growth, although at a rate slower than that of control cells. PARP expression has previously been shown to induce growth retardation in human PARP cDNA-transfected yeast (32,33). mut-PARP remained intact during exposure of cells to staurosporine for 12 h, confirming the resistance of the mutant protein to caspase-3 observed in vitro.…”

Studies of the Expression of Human Poly(ADP-ribose) Polymerase-1 in Saccharomyces cerevisiae and Identification of PARP-1 Substrates by Yeast Proteome Microarray Screening

“…The existence of PAR acceptors in yeast, indicated by our Western blotting analysis and studies conducted by others (16), prompted us to further investigate the identity of these proteins. Toward this goal, we successfully immunoprecipitated some PARlated proteins from yeast extracts using anti-PAR antibody (data not shown).…”

Activation and Caspase-mediated Inhibition of PARP: A Molecular Switch between Fibroblast Necrosis and Apoptosis in Death Receptor Signaling

“…3) NAD ϩ depletion and compromised energy metabolism may alter mitochondrial physiology and cause permeability transition (Hirsch et al, 1997). 4) At least in the yeast system, poly-(ADP-ribose) polymers may impede cell cycle progression because of interference of poly-ADP-ribosylated proteins with chromatin replication (Avila et al, 1994;Collinge and Althaus, 1994). Overall, these mechanisms may be relevant for NO-induced cytotoxicity, excitotoxic damage of neurons, streptozocin-induced diabetes, and reperfusion injury of cardiomyocytes as well as inflammation and other pathological conditions (Heller et al, 1995;Eliasson et al, 1997;Endres et al, 1997;Thiemermann et al, 1997;Ha and Snyder, 1999;Oliver et al, 1999;Bü rkle, 2001;Garcia Soriano et al, 2001).…”