Poly(ADP-ribose)-mediated post-translational modification of chromatin-associated human topoisomerase I. Inhibitory effects on catalytic activity.
We have investigated the association of human topoisomerase I with poly(ADP-ribosylated) domains of chromatin and the effects of this modification on the enzyme activity. In vitro poly(ADP-ribosylation) assays demonstrated that this enzyme was one of the major acceptors for this chromatin-dependent post-translational modification. Western blotting procedures using antibody to topoisomerase I indicated that under extensive poly(ADP-ribosylation) conditions, where a majority of poly(ADP-ribose) acceptor molecules form aggregates, the major population of the topoisomerase I associated with chromatin was apparently non-aggregated. The catalytic activity of the topoisomerase I associated with the poly(ADP-ribosylated) chromatin was 3-5-fold inhibited. Additionally, antibody to poly(ADP-ribose) was used to immunofractionate selectively the modified domains of chromatin. Our data suggests the presence of topoisomerase I, both adjacent and distal to the poly(ADP-ribosylated) sites of chromatin. Unmodified and a significant portion of the modified species of enzyme migrated as approximately 100-kDa proteins. However, the modified form of topoisomerase was noted to be catalytically less active as compared to the enzyme bound to the non-poly(ADP-ribosylated) nucleosomes. These results provide evidence, at the cellular level, for the poly(ADP-ribosylation)-mediated regulation of human topoisomerase I and suggest a functional significance for poly(ADP-ribosylation) in topoisomerase-related processes (replication, transcription, and recombination) in eukaryotes.
Kasid
U N
UN
Department of Biochemistry, Vincent T. Lombardi Cancer Research Center, Georgetown University Medical School, Washington, D.C. 20007.
Halligan
B
B
Liu
L F
LF
Dritschilo
A
A
Smulson
M
M
D015153:Blotting, Western; D002843:Chromatin / Q000201:enzymology*; D002845:Chromatography; D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007118:Immunoassay; D046911:Macromolecular Substances; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D009702:Nucleoside Diphosphate Sugars / Q000378:metabolism*; D011064:Poly Adenosine Diphosphate Ribose / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D059004:Topoisomerase I Inhibitors
D002843:Chromatin; D046911:Macromolecular Substances; D009702:Nucleoside Diphosphate Sugars; D059004:Topoisomerase I Inhibitors; D009243:NAD; D011064:Poly Adenosine Diphosphate Ribose; D004264:DNA Topoisomerases, Type I