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. 1992 Dec;12(12):5673–5682. doi: 10.1128/mcb.12.12.5673

Sequence of cDNA comprising the human pur gene and sequence-specific single-stranded-DNA-binding properties of the encoded protein.

A D Bergemann 1, Z W Ma 1, E M Johnson 1
PMCID: PMC360507  PMID: 1448097

Abstract

The human Pur factor binds strongly to a sequence element repeated within zones of initiation of DNA replication in several eukaryotic cells. The protein binds preferentially to the purine-rich single strand of this element, PUR. We report here the cloning and sequencing of a cDNA encoding a protein with strong affinity for the PUR element. Analysis with a series of mutated oligonucleotides defines a minimal single-stranded DNA Pur-binding element. The expressed Pur open reading frame encodes a protein of 322 amino acids. This protein, Pur alpha, contains three repeats of a consensus motif of 23 amino acids and two repeats of a second consensus motif of 26 amino acids. Near its carboxy terminus, the protein possesses an amphipathic alpha-helix and a glutamine-rich domain. The repeat region of Pur cDNA is homologous to multiple mRNA species in each of several human cell lines and tissues. The HeLa cDNA library also includes a clone encoding a related gene, Pur beta, containing a version of the 23-amino-acid consensus motif similar, but not identical, to those in Pur alpha. Results indicate a novel type of modular protein with capacity to bind repeated elements in single-stranded DNA.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Anachkova B., Hamlin J. L. Replication in the amplified dihydrofolate reductase domain in CHO cells may initiate at two distinct sites, one of which is a repetitive sequence element. Mol Cell Biol. 1989 Feb;9(2):532–540. doi: 10.1128/mcb.9.2.532. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Anderson R. A., Coleman J. E. Physiochemical properties of DNA binding proteins: gene 32 protein of T4 and Escherichia coli unwinding protein. Biochemistry. 1975 Dec 16;14(25):5485–5491. doi: 10.1021/bi00696a017. [DOI] [PubMed] [Google Scholar]
  3. Anderson R. A., Nakashima Y., Coleman J. E. Chemical modifications of functional residues of fd gene 5 DNA-binding protein. Biochemistry. 1975 Mar 11;14(5):907–917. doi: 10.1021/bi00676a006. [DOI] [PubMed] [Google Scholar]
  4. Aviv H., Leder P. Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose. Proc Natl Acad Sci U S A. 1972 Jun;69(6):1408–1412. doi: 10.1073/pnas.69.6.1408. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Benton W. D., Davis R. W. Screening lambdagt recombinant clones by hybridization to single plaques in situ. Science. 1977 Apr 8;196(4286):180–182. doi: 10.1126/science.322279. [DOI] [PubMed] [Google Scholar]
  6. Bergemann A. D., Johnson E. M. The HeLa Pur factor binds single-stranded DNA at a specific element conserved in gene flanking regions and origins of DNA replication. Mol Cell Biol. 1992 Mar;12(3):1257–1265. doi: 10.1128/mcb.12.3.1257. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Brayer G. D., McPherson A. Refined structure of the gene 5 DNA binding protein from bacteriophage fd. J Mol Biol. 1983 Sep 15;169(2):565–596. doi: 10.1016/s0022-2836(83)80065-5. [DOI] [PubMed] [Google Scholar]
  8. Burhans W. C., Vassilev L. T., Caddle M. S., Heintz N. H., DePamphilis M. L. Identification of an origin of bidirectional DNA replication in mammalian chromosomes. Cell. 1990 Sep 7;62(5):955–965. doi: 10.1016/0092-8674(90)90270-o. [DOI] [PubMed] [Google Scholar]
  9. Chen M., Mermod N., Horwitz M. S. Protein-protein interactions between adenovirus DNA polymerase and nuclear factor I mediate formation of the DNA replication preinitiation complex. J Biol Chem. 1990 Oct 25;265(30):18634–18642. [PubMed] [Google Scholar]
  10. Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
  11. Chou P. Y., Fasman G. D. Prediction of protein conformation. Biochemistry. 1974 Jan 15;13(2):222–245. doi: 10.1021/bi00699a002. [DOI] [PubMed] [Google Scholar]
  12. Courey A. J., Holtzman D. A., Jackson S. P., Tjian R. Synergistic activation by the glutamine-rich domains of human transcription factor Sp1. Cell. 1989 Dec 1;59(5):827–836. doi: 10.1016/0092-8674(89)90606-5. [DOI] [PubMed] [Google Scholar]
  13. Dailey L., Caddle M. S., Heintz N., Heintz N. H. Purification of RIP60 and RIP100, mammalian proteins with origin-specific DNA-binding and ATP-dependent DNA helicase activities. Mol Cell Biol. 1990 Dec;10(12):6225–6235. doi: 10.1128/mcb.10.12.6225. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Darnell J. E., Jr Variety in the level of gene control in eukaryotic cells. Nature. 1982 Jun 3;297(5865):365–371. doi: 10.1038/297365a0. [DOI] [PubMed] [Google Scholar]
  15. Eckdahl T. T., Anderson J. N. Conserved DNA structures in origins of replication. Nucleic Acids Res. 1990 Mar 25;18(6):1609–1612. doi: 10.1093/nar/18.6.1609. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Edelmann W., Kröger B., Goller M., Horak I. A recombination hotspot in the LTR of a mouse retrotransposon identified in an in vitro system. Cell. 1989 Jun 16;57(6):937–946. doi: 10.1016/0092-8674(89)90332-2. [DOI] [PubMed] [Google Scholar]
  17. Erdile L. F., Heyer W. D., Kolodner R., Kelly T. J. Characterization of a cDNA encoding the 70-kDa single-stranded DNA-binding subunit of human replication protein A and the role of the protein in DNA replication. J Biol Chem. 1991 Jun 25;266(18):12090–12098. [PubMed] [Google Scholar]
  18. Feinberg A. P., Vogelstein B. "A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity". Addendum. Anal Biochem. 1984 Feb;137(1):266–267. doi: 10.1016/0003-2697(84)90381-6. [DOI] [PubMed] [Google Scholar]
  19. Flavin M., Strauss F. Multiple sequence-specific single-strand-binding proteins for the promoter region of the rat albumin gene. DNA Cell Biol. 1991 Mar;10(2):113–118. doi: 10.1089/dna.1991.10.113. [DOI] [PubMed] [Google Scholar]
  20. Frohman M. A., Dush M. K., Martin G. R. Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc Natl Acad Sci U S A. 1988 Dec;85(23):8998–9002. doi: 10.1073/pnas.85.23.8998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Gaillard C., Weber M., Strauss F. A sequence-specific single-strand-binding protein for the late-coding strand of the simian virus 40 control region. J Virol. 1988 Jul;62(7):2380–2385. doi: 10.1128/jvi.62.7.2380-2385.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Ge H., Zuo P., Manley J. L. Primary structure of the human splicing factor ASF reveals similarities with Drosophila regulators. Cell. 1991 Jul 26;66(2):373–382. doi: 10.1016/0092-8674(91)90626-a. [DOI] [PubMed] [Google Scholar]
  23. Gutiérrez C., Martín G., Sogo J. M., Salas M. Mechanism of stimulation of DNA replication by bacteriophage phi 29 single-stranded DNA-binding protein p5. J Biol Chem. 1991 Feb 5;266(4):2104–2111. [PubMed] [Google Scholar]
  24. Handeli S., Klar A., Meuth M., Cedar H. Mapping replication units in animal cells. Cell. 1989 Jun 16;57(6):909–920. doi: 10.1016/0092-8674(89)90329-2. [DOI] [PubMed] [Google Scholar]
  25. Hay N., Bishop J. M., Levens D. Regulatory elements that modulate expression of human c-myc. Genes Dev. 1987 Sep;1(7):659–671. doi: 10.1101/gad.1.7.659. [DOI] [PubMed] [Google Scholar]
  26. Haynes S. R., Raychaudhuri G., Beyer A. L. The Drosophila Hrb98DE locus encodes four protein isoforms homologous to the A1 protein of mammalian heterogeneous nuclear ribonucleoprotein complexes. Mol Cell Biol. 1990 Jan;10(1):316–323. doi: 10.1128/mcb.10.1.316. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Haynes S. R., Rebbert M. L., Mozer B. A., Forquignon F., Dawid I. B. pen repeat sequences are GGN clusters and encode a glycine-rich domain in a Drosophila cDNA homologous to the rat helix destabilizing protein. Proc Natl Acad Sci U S A. 1987 Apr;84(7):1819–1823. doi: 10.1073/pnas.84.7.1819. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Heintz N. H., Hamlin J. L. An amplified chromosomal sequence that includes the gene for dihydrofolate reductase initiates replication within specific restriction fragments. Proc Natl Acad Sci U S A. 1982 Jul;79(13):4083–4087. doi: 10.1073/pnas.79.13.4083. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Hofmann J. F., Gasser S. M. Identification and purification of a protein that binds the yeast ARS consensus sequence. Cell. 1991 Mar 8;64(5):951–960. doi: 10.1016/0092-8674(91)90319-t. [DOI] [PubMed] [Google Scholar]
  30. Hope I. A., Struhl K. Functional dissection of a eukaryotic transcriptional activator protein, GCN4 of yeast. Cell. 1986 Sep 12;46(6):885–894. doi: 10.1016/0092-8674(86)90070-x. [DOI] [PubMed] [Google Scholar]
  31. Jeffreys A. J., Wilson V., Thein S. L. Individual-specific 'fingerprints' of human DNA. Nature. 1985 Jul 4;316(6023):76–79. doi: 10.1038/316076a0. [DOI] [PubMed] [Google Scholar]
  32. Kennelly P. J., Krebs E. G. Consensus sequences as substrate specificity determinants for protein kinases and protein phosphatases. J Biol Chem. 1991 Aug 25;266(24):15555–15558. [PubMed] [Google Scholar]
  33. Kozak M. Compilation and analysis of sequences upstream from the translational start site in eukaryotic mRNAs. Nucleic Acids Res. 1984 Jan 25;12(2):857–872. doi: 10.1093/nar/12.2.857. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Krowczynska A. M., Rudders R. A., Krontiris T. G. The human minisatellite consensus at breakpoints of oncogene translocations. Nucleic Acids Res. 1990 Mar 11;18(5):1121–1127. doi: 10.1093/nar/18.5.1121. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Leffak M., James C. D. Opposite replication polarity of the germ line c-myc gene in HeLa cells compared with that of two Burkitt lymphoma cell lines. Mol Cell Biol. 1989 Feb;9(2):586–593. doi: 10.1128/mcb.9.2.586. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Levin J. M., Robson B., Garnier J. An algorithm for secondary structure determination in proteins based on sequence similarity. FEBS Lett. 1986 Sep 15;205(2):303–308. doi: 10.1016/0014-5793(86)80917-6. [DOI] [PubMed] [Google Scholar]
  37. Maeda K., Kneale G. G., Tsugita A., Short N. J., Perham R. N., Hill D. F., Petersen G. B. The DNA-binding protein of Pf1 filamentous bacteriophage: amino-acid sequence and structure of the gene. EMBO J. 1982;1(2):255–261. doi: 10.1002/j.1460-2075.1982.tb01156.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Marahrens Y., Stillman B. A yeast chromosomal origin of DNA replication defined by multiple functional elements. Science. 1992 Feb 14;255(5046):817–823. doi: 10.1126/science.1536007. [DOI] [PubMed] [Google Scholar]
  39. Neale G. A., Kitchingman G. R. Biochemical analysis of adenovirus type 5 DNA-binding protein mutants. J Biol Chem. 1989 Feb 25;264(6):3153–3159. [PubMed] [Google Scholar]
  40. Plyte S. E., Kneale G. G. The role of tyrosine residues in the DNA-binding site of the Pf1 gene 5 protein. Protein Eng. 1991 Jun;4(5):553–560. doi: 10.1093/protein/4.5.553. [DOI] [PubMed] [Google Scholar]
  41. Prasad B. V., Chiu W. Sequence comparison of single-stranded DNA binding proteins and its structural implications. J Mol Biol. 1987 Feb 5;193(3):579–584. doi: 10.1016/0022-2836(87)90268-3. [DOI] [PubMed] [Google Scholar]
  42. Ptashne M. How eukaryotic transcriptional activators work. Nature. 1988 Oct 20;335(6192):683–689. doi: 10.1038/335683a0. [DOI] [PubMed] [Google Scholar]
  43. Quinn C. O., Kitchingman G. R. Functional analysis of the adenovirus type 5 DNA-binding protein: site-directed mutants which are defective for adeno-associated virus helper activity. J Virol. 1986 Nov;60(2):653–661. doi: 10.1128/jvi.60.2.653-661.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Rhode P. R., Elsasser S., Campbell J. L. Role of multifunctional autonomously replicating sequence binding factor 1 in the initiation of DNA replication and transcriptional control in Saccharomyces cerevisiae. Mol Cell Biol. 1992 Mar;12(3):1064–1077. doi: 10.1128/mcb.12.3.1064. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Rogers S., Wells R., Rechsteiner M. Amino acid sequences common to rapidly degraded proteins: the PEST hypothesis. Science. 1986 Oct 17;234(4774):364–368. doi: 10.1126/science.2876518. [DOI] [PubMed] [Google Scholar]
  46. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Singh H., LeBowitz J. H., Baldwin A. S., Jr, Sharp P. A. Molecular cloning of an enhancer binding protein: isolation by screening of an expression library with a recognition site DNA. Cell. 1988 Feb 12;52(3):415–423. doi: 10.1016/s0092-8674(88)80034-5. [DOI] [PubMed] [Google Scholar]
  48. Steinert P. M., Mack J. W., Korge B. P., Gan S. Q., Haynes S. R., Steven A. C. Glycine loops in proteins: their occurrence in certain intermediate filament chains, loricrins and single-stranded RNA binding proteins. Int J Biol Macromol. 1991 Jun;13(3):130–139. doi: 10.1016/0141-8130(91)90037-u. [DOI] [PubMed] [Google Scholar]
  49. Vassilev L., Johnson E. M. An initiation zone of chromosomal DNA replication located upstream of the c-myc gene in proliferating HeLa cells. Mol Cell Biol. 1990 Sep;10(9):4899–4904. doi: 10.1128/mcb.10.9.4899. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Vaughn J. P., Dijkwel P. A., Hamlin J. L. Replication initiates in a broad zone in the amplified CHO dihydrofolate reductase domain. Cell. 1990 Jun 15;61(6):1075–1087. doi: 10.1016/0092-8674(90)90071-l. [DOI] [PubMed] [Google Scholar]
  51. Wang Y. S., Hall J. D. Characterization of a major DNA-binding domain in the herpes simplex virus type 1 DNA-binding protein (ICP8). J Virol. 1990 May;64(5):2082–2089. doi: 10.1128/jvi.64.5.2082-2089.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Wobbe C. R., Weissbach L., Borowiec J. A., Dean F. B., Murakami Y., Bullock P., Hurwitz J. Replication of simian virus 40 origin-containing DNA in vitro with purified proteins. Proc Natl Acad Sci U S A. 1987 Apr;84(7):1834–1838. doi: 10.1073/pnas.84.7.1834. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. de Jong E. A., van Duynhoven J. P., Harmsen B. J., Konings R. N., Hilbers C. W. Two-dimensional 1H nuclear magnetic resonance studies on the gene V-encoded single-stranded DNA-binding protein of the filamentous bacteriophage IKe. I. Structure elucidation of the DNA-binding wing. J Mol Biol. 1989 Mar 5;206(1):119–132. doi: 10.1016/0022-2836(89)90528-7. [DOI] [PubMed] [Google Scholar]
  54. van Duynhoven J. P., Folkers P. J., Stassen A. P., Harmsen B. J., Konings R. N., Hilbers C. W. Structure of the DNA binding wing of the gene-V encoded single- stranded DNA binding protein of the filamentous bacteriophage M13. FEBS Lett. 1990 Feb 12;261(1):1–4. doi: 10.1016/0014-5793(90)80621-o. [DOI] [PubMed] [Google Scholar]

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