FUNCTIONAL EXPRESSION OF HUMAN POLY(ADP-RIBOSE) POLYMERASE IN SCHIZOSACCHAROMYCES-POMBE RESULTS IN MITOTIC DELAY AT G(1), INCREASED MUTATION-RATE, AND SENSITIZATION TO RADIATION

The activity of poly(ADP-ribose) polymerase (PADPRP), a chromatin-asso ciated enzyme present in most eukaryotic cells, is stimulated by DNA s trand breaks, suggesting a role for the enzyme in the cellular respons e to DNA damage. However, the primary function of PADPRP remains unkno wn. We have selected Schizosaccharomyces pombe as a simple eukaryotic system in which to study PADPRP function because this fission yeast sh ares with mammalian cells important cellular features possibly associa ted with poly-(ADP-ribos)ylation pathways. We investigated the existen ce of an endogenous yeast PADPRP by DNA and RNA hybridization to mamma lian probes under low-stringency conditions and by PADPRP activity ass ays. Our data indicate that fission yeasts are naturally devoid of PAD PRP. We therefore isolated S. pombe strains expressing PADPRP by trans formation with a human full-length PADPRP cDNA under the control of th e 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 polypeptid e, identical in size to the native human PADPRP protein. Yeast recombi nant 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 retardat ion as a result of a specific delay at the G(1) phase, (ii) decreased cell viability in stationary cultures, (iii) enhanced rates of spontan eous and radiation-induced ade6-ade7 mutations, and (iv) increased sen sitivity to radiation. SPT strains may prove efficient tools with whic h to investigate PADPRP functions in eukaryotic cells.