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cDNA in COS cells was also shown to increase the ability ofthese cells to repair DNA lesions caused by y radiation (unpublished data).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...

cDNAs encoding poly(ADP-ribose) polymerase from a human hepatoma lambda gt11 cDNA library were isolated by immunological screening. One insert of 1.3 kilobases (kb) consistently hybridized on RNA gel blots to an mRNA species of 3.6-3.7 kb, which is consistent with the size of RNA necessary to code for the polymerase protein (116 kDa). This insert was subsequently used in both in vitro hybrid selection and hybrid-arrested translation studies. An mRNA species from HeLa cells of 3.6-3.7 kb was selected that was translated into a 116-kDa protein, which was selectively immunoprecipitated with anti-poly (ADP-ribose) polymerase. To confirm that the 1.3-kb insert from lambda gt11 encodes for poly(ADP-ribose) polymerase, the insert was used to screen a 3- to 4-kb subset of a transformed human fibroblast cDNA library in the Okayama-Berg vector. One of these vectors [pcD-p(ADPR)P; 3.6 kb] was tested in transient transfection experiments in COS cells. This cDNA insert contained the complete coding sequence for polymerase as indicated by the following criteria: A 3-fold increase in in vitro activity was noted in extracts from transfected cells compared to mock or pSV2-CAT transfected cells. A 6-fold increase in polymerase activity in pcD-p(ADPR)P transfected cell extracts compared to controls was observed by "activity gel" analysis on gels of electrophoretically separated proteins at 116 kDa. A 10- to 15-fold increase in newly synthesized polymerase was detected by immunoprecipitation of labeled transfected cell extracts. Using pcD-p(ADPR)P as probe, it was observed that the level of poly(ADP-ribose) polymerase mRNA was elevated at 5 and 7 hr of S phase of the HeLa cell cycle, but was unaltered when artificial DNA strand breaks are introduced in HeLa cells by alkylating agents.

No abstract

No abstract