cDNA sequence, protein structure, and chromosomal location of the human gene for poly(ADP-ribose) polymerase.

Abstract

Recently we described a full-length cDNA for the human nuclear enzyme poly(ADP-ribose) polymerase. Here, we report the chromosomal localization and partial map of the human gene for this enzyme as well as the complete coding sequence for this protein. The nucleotide sequence reveals a single 3042-base open reading frame encoding a protein with a predicted Mr of 113,135. A comparison of this deduced amino acid sequence with the amino acid sequence of three peptides derived from human poly(ADP-ribose) polymerase revealed a match of 27 amino acid residues. A computerderived structural analysis of the enzyme and a search for similarities with other proteins confirmed that the polymerase belongs to a subfamily of DNA/NAD-binding proteins and DNA-repair proteins. Possible Zn2+-binding "fingers," a nucleotide-binding fold, and a nuclear transport signal were noted. Additionally, chromosomal mapping has identified polymerase-hybridizing sequences on human chromosomes 1 (the active gene), 13, and 14 (processed pseudogenes). Using the polymerase cDNA as a probe, we also have detected several DNA restriction fragment length polymorphisms in normal humans.Considerable work in the past few years has centered on the possible role of the nuclear enzyme, poly(ADP-ribose) polymerase, in modulating the DNA replication/repair processes of mammalian cells (1). This enzyme uses NAD as a substrate in the formation of poly(ADP-ribose) chains at sites on many nuclear proteins. The enzyme binds tightly to DNA and requires DNA strand breaks for enzymatic activity (2). Since cells carrying a mutated polymerase are not yet available, a reasonable approach to answering questions about the biochemical and biological functions of this protein is through gene cloning. We have described (3) the cloning of poly(ADPribose) polymerase cDNA from a phage Xgtll library. This 1.3-kilobase (kb) insert was used in both hybrid-selection and arrest studies to confirm the identity of the partial cDNA. Subsequently, a 3.7-kb full-length cDNA contained in the Okayama-Berg expression vector was isolated, and transfection with this recombinant plasmid caused significant levels of transient polymerase expression in COS cells (3). The hyperexpression of this cloned cDNA in COS cells was also shown to increase the ability ofthese cells to repair DNA lesions caused by y radiation (unpublished data). These results suggest that this gene encodes a protein that is functionally involved with DNA repair in eukaryotic cells. A partial polymerase cDNA clone has recently been reported by Suzuki et al. (5).The enzyme is composed of three functionally distinct regions, an NH2-terminal DNA-binding domain, a central automodification domain, and a COOH-terminal NAD-binding region (6). We now report the DNA § and amino acid sequences of this enzyme and a structural analysis of its domains.We also used various regions of the poly(ADP-ribose) polymerase cDNA as probes to chromosomally map the human gene for this nuclear enzyme by Southern analysis of DNAs isolated from hu...

PARP-1 binds E2F-1 independently of its DNA binding and catalytic domains, and acts as a novel coactivator of E2F-1-mediated transcription during re-entry of quiescent cells into S phase

“…Reprobing of the immunoblot with antibodies to E2F-1 revealed a B60-kDa band, distinct from the 4114 kDa band detected with anti-PAR (Figure 2 right panel), thus, indicating that the modified protein was not E2F-1. PARP-1 is the main acceptor in these reactions, attributable to the presence of up to 28 automodification sites in the protein (Kawaichi et al, 1981;Cherney et al, 1987;Desmarais et al, 1991). In contrast to E2F-1, we have demonstrated that GST-p53 is an effective substrate for PARP-1 under the same conditions (Simbulan- Rosenthal et al, 2001).…”

“…PARP-1, the most abundant and most extensively studied member of this family, is activated allosterically by binding to DNA ends and plays essential roles in various nuclear processes Menissier-de Murcia et al, 1989). PARP-1 undergoes extensive autopoly(ADP-ribosyl)ation in a central automodification domain (Kawaichi et al, 1981;Cherney et al, 1987). PARP-2 and PARP-3 (Johansson, 1999) apparently account for the residual PARP activity in PARP-1-deficient cells (Shieh et al, 1998;Ame et al, 1999).…”

Identification of novel components of NAD-utilizing metabolic pathways and prediction of their biochemical functions

“…38 In bacteria, they are central to the reversal of the acyl modifications of enzymes such as propionyl-CoA synthetase, which generates the substrate for the acyl modification in the first place. 39 Both the free and the ligated ADP-ribose moieties and its various derivatives serve as targets for recognition by specialized binding domains such as the PARP finger, a treble clef domain that binds polyADP ribose, 40,41 and the Macro 42 and inactive Nudix domains. 38 Some Macro domains also function as enzymes that hydrolyze ADP ribose derivatives, such as polyADP ribose chains 43 or OAADPR.…”

DNA transitions induced by binding of PARP‐1 to cruciform structures in supercoiled plasmids

“…PARP-1 has been implicated in the DNA damage response as well as in cell growth, regulation, and apoptosis (2)(3)(4). The structure of the 113 kDa PARP-1 protein consists of three functional domains: the DNA binding domain, the central automodification domain, and the catalytic domain (5,6). The N-terminal DNA binding domain (DBD) contains two zinc fingers that recognize and bind to single and double-stranded DNA breaks (7,8).…”

“…However, PARP-1 can also bind to supercoiled DNA (9), cruciform structures (10), DNA loops (9), and base unpaired regions (11). The central automodification domain is an acceptor of ADP-ribose polymers, and the N-terminus is part of the automodification domain containing a leucine-zipper motif that may be responsible for protein-protein interactions (12,6,13). A catalytic domain is located in the C-terminal fragment, and catalyzes the formation of the branched ADP-ribose polymers from NAD 1 (6,14).…”