Expression and characterization of a fusion protein between the catalytic domain of poly(ADP-ribose) polymerase and the DNA binding domain of the glucocorticoid receptor.
A fusion protein comprising the DNA-binding region of the glucocorticoid receptor and the catalytic domain of poly(ADP-ribose) polymerase was constructed. This chimeric protein was expressed both in E. coli and in eukaryotic cells and was recognized by antibodies to both polymerase and the glucocorticoid receptor. Similar to polymerase, the chimera produced bona fide poly (ADP-ribose) polymers covalently bound to protein and was inhibited by 3-aminobenzamide. Like the authentic glucocorticoid receptor, the fusion protein formed a stable complex with DNA containing the glucocorticoid response element. In mammalian cells, the fusion protein significantly and specifically inhibited the ability of the glucocorticoid receptor to stimulate a reporter construct. These results indicate that polymerase activity can be targeted to specific DNA sequences and modulate gene expression.
Rosenthal
D
D
Department of Biochemistry and Molecular Biology, Georgetown University School of Medicine, Washington, DC 20007.
Hong
T
T
Cherney
B
B
Zhang
S
S
Shima
T
T
Danielsen
M
M
Smulson
M
M
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000818:Animals; D001549:Benzamides / Q000494:pharmacology; D001665:Binding Sites; D002384:Catalysis; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D015151:Immunoblotting; D007163:Immunosorbent Techniques; D010957:Plasmids; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D011065:Poly(ADP-ribose) Polymerases / Q000235:genetics* / Q000378:metabolism; D011965:Receptors, Glucocorticoid / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics*; D014162:Transfection
D001549:Benzamides; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D011965:Receptors, Glucocorticoid; D011993:Recombinant Fusion Proteins; D000246:Adenosine Diphosphate Ribose; C025160:3-aminobenzamide; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase; D011065:Poly(ADP-ribose) Polymerases