Functional expression of human poly(ADP-ribose) polymerase in Schizosaccharomyces pombe results in mitotic delay at G1, increased mutation rate, and sensitization to radiation.
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.
Avila
M A
MA
Department of Radiation Medicine, Georgetown University Medical Center, Washington, DC 20007.
Velasco
J A
JA
Smulson
M E
ME
Dritschilo
A
A
Castro
R
R
Notario
V
V
D004307:Dose-Response Relationship, Radiation; D016193:G1 Phase / Q000502:physiology*; D005720:Gamma Rays / Q000009:adverse effects; D006801:Humans; D016296:Mutagenesis / Q000502:physiology*; D011065:Poly(ADP-ribose) Polymerases / Q000096:biosynthesis* / Q000235:genetics; D011836:Radiation Tolerance / Q000502:physiology*; D011994:Recombinant Proteins / Q000096:biosynthesis; D012568:Schizosaccharomyces / Q000235:genetics / Q000502:physiology*; D014170:Transformation, Genetic
D011994:Recombinant Proteins; D011065:Poly(ADP-ribose) Polymerases