title: Depletion of poly(ADP-ribose) polymerase by antisense RNA expression results in a delay in DNA strand break rejoining.
abstract: The effects of inducible expression of poly(ADP-ribose) polymerase (PADPRP) antisense RNA in HeLa cells were determined in order to gain further insight into the biological roles of the poly(ADP-ribosyl)ation modification of nuclear proteins. A recombinant expression plasmid was prepared with the mouse mammary tumor virus (MMTV) promoter upstream of the antisense-oriented PADPRP cDNA. Expression of the antisense RNA was under strict control, with negligible effects on cell growth being apparent in the absence of inducer. Consistent with the previously described stability of PADPRP (half-life of at least 2 days, in vivo), 48-72 h were required after induction of antisense RNA expression by dexamethasone for the abundant concentration of PADPRP, normally present in HeLa cells, to be reduced by greater than 80%. The depletion of endogenous PADPRP as mediated by induced antisense RNA expression was established by: (i) a progressive synthesis of antisense transcripts in cells as assessed by Northern analysis; (ii) an 80% decrease in activity of the enzyme; and (iii) a greater than 90% reduction in the cellular content of PADPRP protein, as demonstrated by both immunoblotting and immunohistochemical analysis in intact cells. Several biological parameters were monitored in cells depleted of PADPRP. The chromatin of PADPRP-depleted cells was shown to have an altered structure as assessed by deoxyribonuclease I susceptibility. Cell morphology was also altered, with multinucleated aggregates being evident 72 h after induction of antisense RNA expression. Cells depleted of PADPRP were not able to commence DNA strand break joining of damaged DNA. However, DNA repair capacity was re-established at later time periods, indicating that PADPRP may contribute to alterations in chromatin structure that occur initially in DNA strand break rejoining and that the concentration of the enzyme in nuclei exceeds the requirement for DNA repair/replication.
authors.affiliation: Department of Biochemistry and Molecular Biology, Georgetown University, Washington, D.C. 20007.
authors.forename: R
authors.initials: R
authors.lastname: Ding
authors.forename: Y
authors.initials: Y
authors.lastname: Pommier
authors.forename: V H
authors.initials: VH
authors.lastname: Kang
authors.forename: M
authors.initials: M
authors.lastname: Smulson
mesh_terms: D003001:Cloning, Molecular; D004249:DNA Damage; D004260:DNA Repair; D003850:Deoxyribonuclease I / Q000378:metabolism; D003907:Dexamethasone / Q000494:pharmacology; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007150:Immunohistochemistry; D007700:Kinetics; D010641:Phenotype; D011065:Poly(ADP-ribose) Polymerases / Q000235:genetics / Q000378:metabolism*; D016372:RNA, Antisense / Q000378:metabolism*; D014162:Transfection
chemical_list: D016372:RNA, Antisense; D003907:Dexamethasone; D011065:Poly(ADP-ribose) Polymerases; D003850:Deoxyribonuclease I