The carboxyl-terminal domain of human poly(ADP-ribose) polymerase. Overproduction in Escherichia coli, large scale purification, and characterization.
The cDNA encoding the carboxyl-terminal 40-kDa domain of human poly(ADP-ribose) polymerase was inserted into an expression vector. The recombinant protein was overproduced in Escherichia coli, and purified to homogeneity. The 40-kDa domain had the same affinity (Km) for NAD+ as the full-length enzyme, expressed abortive NAD+ glycohydrolase activity, catalyzed the initiation, elongation, and branching of ADP-ribose polymers, but exhibited no DNA dependence. Its specific activity was approximately 500-fold lower than that of the whole enzyme activated by DNA strand breaks. Surprisingly, the carboxyl-terminal 40-kDa domain exhibited the processive mode of polymer attachment typical of full-length poly(ADP-ribose) polymerase and was able to modify histones H1 and H2B. Finally, the polymer sizes formed by the 40-kDa domain were influenced by histone H1.
Institut de Biologie Moléculaire et Cellulaire du Centre National de la Recherche Scientifique, Strasbourg, France.
F
F
Simonin
L
L
Höfferer
P L
PL
Panzeter
S
S
Muller
G
G
de Murcia
F R
FR
Althaus
D001483:Base Sequence; D002384:Catalysis; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D006801:Humans; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism; D009244:NAD+ Nucleosidase / Q000378:metabolism; D011065:Poly(ADP-ribose) Polymerases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D009243:NAD; D004247:DNA; D011065:Poly(ADP-ribose) Polymerases; D009244:NAD+ Nucleosidase