M. Masutani et al., CLONING AND FUNCTIONAL EXPRESSION OF POLY(ADP-RIBOSE) POLYMERASE CDNAFROM SARCOPHAGA-PEREGRINA, European journal of biochemistry, 220(2), 1994, pp. 607-614
A cDNA spanning the entire coding region for poly(ADP-ribose) polymera
se (PARP) of Sarcophaga peregrina was isolated and the nucleotide sequ
ence was determined. The longest open reading frame encodes a polypept
ide of 996 amino acid residues with a molecular mass of 113 033 Da. Th
e similarities to the human PARP in amino acid sequence were relativel
y low in the DNA-binding and auto-modification domains, but very high
in the C-terminal catalytic domain: identity of amino acids is 34% in
the N-terminal DNA-binding domain (residues 1-369), 27% in the auto-mo
dification domain (residues 370-507), and 56% in the C-terminal NAD-bi
nding domain (residues 508-996). Two zinc-fingers (C-X(2)-C-X(28)-H-X(
2)-C and C-X(2)-C-X(31)-H-X(2)-C)(2) and a basic region in the N-termi
nal DNA-binding domain recognized in other PARR are conserved. Downstr
eam of the basic region, another cysteine-rich motif (C-X(2)-C-X(13)-C
-X(9)-C), a putative zinc-finger, was found to be well conserved in th
e PARP of Sarcophaga, Drosophila and human. A leucine-zipper motif (L-
X(6)L-X(6)-L-X(6)-L) which was found in the auto-modification domain o
f Drosophila PARP, is disrupted in the Sarcophaga enzyme: the second l
eucine is replaced by proline, and the third leucine by valine. Full-l
ength cDNA for Sarcophaga PARP was cloned into an expression plasmid a
nd expressed in Escherichia coli. A lysate of E. coli cells containing
expressed protein reacted with antibody against Sarcophaga PARP, and
PARP activity was detected. Thus, we conclude that isolated cDNA encod
es a functional Sarcophaga PARP cDNA.