The 40 kDa carboxy-terminal domain of poly(ADP-ribose) polymerase-1 forms catalytically competent homo- and heterodimers in the absence of DNA.
The 40 kDa carboxy-terminal catalytic domain (CD) of avian poly(ADP-ribose) polymerase (PARP-1) was cloned, expressed in a baculovirus expression system, and purified to homogeneity by affinity chromatography. The purified polypeptide synthesized covalent CD-poly(ADP-ribose) conjugates in the absence of DNA. Electrophoretic analysis of the ADP-ribose chain length distribution generated indicated that recombinant CD was able to catalyze the initiation, elongation, and branching reactions of poly(ADP-ribose) synthesis, although at a 500-fold lower efficiency than wild-type PARP-1. Kinetic evaluation of poly(ADP-ribose) synthesis showed that the enzymatic activities of CD increased for up to 60 minutes in a time-dependent manner. Moreover, the rates of CD auto-poly(ADP-ribosyl)ation increased with second-order kinetics as a function of the protein concentration with either betaNAD(+) or 3'-deoxyNAD(+) as a substrate. Furthermore, the formation of catalytically competent CD-[PARP-1] heterodimers was also observed in specific ultrafiltration experiments. Thus, we conclude that the 40 kDa carboxy terminus of PARP-1 forms a competent catalytic dimer in the absence of DNA, and that its automodification reaction is intermolecular.
Mendoza-Alvarez
Hilda
H
The Department of Molecular Biology and Immunology, University of North Texas, Health Science Center at Fort Worth, 3500 Camp Bowie Boulevard, Fort Worth, TX 76107-2699, USA.
Alvarez-Gonzalez
Rafael
R
D000818:Animals; D020134:Catalytic Domain; D002645:Chickens; D004247:DNA / Q000378:metabolism*; D019281:Dimerization; D006801:Humans; D009243:NAD / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011065:Poly(ADP-ribose) Polymerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D010446:Peptide Fragments; D011994:Recombinant Proteins; D009243:NAD; D004247:DNA; D011065:Poly(ADP-ribose) Polymerases