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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1987 Mar;84(5):1224–1228. doi: 10.1073/pnas.84.5.1224

Cloning and expression of cDNA for human poly(ADP-ribose) polymerase.

H M Alkhatib, D F Chen, B Cherney, K Bhatia, V Notario, C Giri, G Stein, E Slattery, R G Roeder, M E Smulson
PMCID: PMC304399  PMID: 3029772

Abstract

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.

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Selected References

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  1. Benjamin R. C., Gill D. M. Poly(ADP-ribose) synthesis in vitro programmed by damaged DNA. A comparison of DNA molecules containing different types of strand breaks. J Biol Chem. 1980 Nov 10;255(21):10502–10508. [PubMed] [Google Scholar]
  2. Berger N. A., Sikorski G. W., Petzold S. J., Kurohara K. K. Defective poly(adenosine diphosphoribose) synthesis in xeroderma pigmentosum. Biochemistry. 1980 Jan 22;19(2):289–293. doi: 10.1021/bi00543a006. [DOI] [PubMed] [Google Scholar]
  3. Cherney B. W., Midura R. J., Caplan A. I. Poly(ADP-ribose) synthetase and chick limb mesenchymal cell differentiation. Dev Biol. 1985 Nov;112(1):115–125. doi: 10.1016/0012-1606(85)90125-3. [DOI] [PubMed] [Google Scholar]
  4. Fagan J. B., Pastewka J. V., Park S. S., Guengerich F. P., Gelboin H. V. Identification and quantitation of a 2.0-kilobase messenger ribonucleic acid coding for 3-methylcholanthrene-induced cytochrome P-450 using cloned cytochrome P-450 complementary deoxyribonucleic acid. Biochemistry. 1982 Dec 7;21(25):6574–6580. doi: 10.1021/bi00268a039. [DOI] [PubMed] [Google Scholar]
  5. Juarez-Salinas H., Sims J. L., Jacobson M. K. Poly(ADP-ribose) levels in carcinogen-treated cells. Nature. 1979 Dec 13;282(5740):740–741. doi: 10.1038/282740a0. [DOI] [PubMed] [Google Scholar]
  6. Jump D. B., Butt T. R., Smulson M. Nuclear protein modification and chromatin substructure. 3. Relationship between poly(adenosine diphosphate) ribosylation and different functional forms of chromatin. Biochemistry. 1979 Mar 20;18(6):983–990. doi: 10.1021/bi00573a008. [DOI] [PubMed] [Google Scholar]
  7. Kidwell W. R., Mage M. G. Changes in poly(adenosine diphosphate-ribose) and poly(adenosine diphosphate-ribose) polymerase in synchronous HeLa cells. Biochemistry. 1976 Mar 23;15(6):1213–1217. doi: 10.1021/bi00651a006. [DOI] [PubMed] [Google Scholar]
  8. Malik N., Bustin M., Smulson M. Antibody to poly(adenosine diphosphate-ribose) polymerase and its use in chromatin analysis. Nucleic Acids Res. 1982 May 11;10(9):2939–2950. doi: 10.1093/nar/10.9.2939. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Malik N., Miwa M., Sugimura T., Thraves P., Smulson M. Immunoaffinity fractionation of the poly(ADP-ribosyl)ated domains of chromatin. Proc Natl Acad Sci U S A. 1983 May;80(9):2554–2558. doi: 10.1073/pnas.80.9.2554. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Miller J. S., Paterson B. M., Ricciardi R. P., Cohen L., Roberts B. E. Methods utilizing cell-free protein-synthesizing systems for the identification of recombinant DNA molecules. Methods Enzymol. 1983;101:650–674. doi: 10.1016/0076-6879(83)01046-0. [DOI] [PubMed] [Google Scholar]
  11. Okayama H., Berg P. A cDNA cloning vector that permits expression of cDNA inserts in mammalian cells. Mol Cell Biol. 1983 Feb;3(2):280–289. doi: 10.1128/mcb.3.2.280. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Pardee A. B., Boorstein R. J., Lau C. C. Interference with DNA repair mechanisms of mammalian cells: cell cycle dependence. Princess Takamatsu Symp. 1983;13:287–294. [PubMed] [Google Scholar]
  13. Scovassi A. I., Stefanini M., Bertazzoni U. Catalytic activities of human poly(ADP-ribose) polymerase from normal and mutagenized cells detected after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem. 1984 Sep 10;259(17):10973–10977. [PubMed] [Google Scholar]
  14. Slattery E., Dignam J. D., Matsui T., Roeder R. G. Purification and analysis of a factor which suppresses nick-induced transcription by RNA polymerase II and its identity with poly(ADP-ribose) polymerase. J Biol Chem. 1983 May 10;258(9):5955–5959. [PubMed] [Google Scholar]
  15. Smulson M. E., Schein P., Mullins D. W., Jr, Sudhakar S. A putative role for nicotinamide adenine dinucleotide-promoted nuclear protein modification in the antitumor activity of N-methyl-N-nitrosourea. Cancer Res. 1977 Sep;37(9):3006–3012. [PubMed] [Google Scholar]
  16. Stein G. S., Borun T. W. The synthesis of acidic chromosomal proteins during the cell cycle of HeLa S-3 cells. I. The accelerated accumulation of acidic residual nuclear protein before the initiation of DNA replication. J Cell Biol. 1972 Feb;52(2):292–307. doi: 10.1083/jcb.52.2.292. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Thraves P. J., Kasid U., Smulson M. E. Selective isolation of domains of chromatin proximal to both carcinogen-induced DNA damage and poly-adenosine diphosphate-ribosylation. Cancer Res. 1985 Jan;45(1):386–391. [PubMed] [Google Scholar]
  18. Wong M., Kanai Y., Miwa M., Bustin M., Smulson M. Immunological evidence for the in vivo occurrence of a crosslinked complex of poly(ADP-ribosylated) histone H1. Proc Natl Acad Sci U S A. 1983 Jan;80(1):205–209. doi: 10.1073/pnas.80.1.205. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Young R. A., Davis R. W. Efficient isolation of genes by using antibody probes. Proc Natl Acad Sci U S A. 1983 Mar;80(5):1194–1198. doi: 10.1073/pnas.80.5.1194. [DOI] [PMC free article] [PubMed] [Google Scholar]

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