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1999 Fiscal Year Final Research Report Summary

DNA binding mechanism of nuclear matrix proteins that recognize short AT-tracts

Research Project

Project/Area Number 09680592
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Structural biochemistry
Research InstitutionOkayama University

Principal Investigator

TSUTSUKI Ken  Okayama University, Medical School, Assistant Professor, 医学部, 助教授 (70108158)

Project Period (FY) 1997 – 1999
KeywordsDNA binding protein / AT-tracts / Nuclear matrix / Nuclear scaffold / MAR / SAR
Research Abstract

SP120 is a major nuclear matrix protein that binds specifically to the matrix attachment region DNA (MAR) to organize the genome into domains. To delineate sequence requirements for the recognition by SP120, we used synthetic concatemers as an artificial MAR. The protein showed a high affinity to the concatemer synthesized by ligation of a duplex monomer (26 nt) with 4-bp overhangs. The monomer duplex contained two short homopolymeric AT-tracts although the overall AT content was only 42%. Orientation of the monomer units in the concatemer is randomized because of the palindromic overhang and this type of concatemer was referred to as "RC" for random concatemer. Other types of concatemers ("DC" and "AC" representing direct and alternating orientations, respectively) were also synthesized by manipulating the overhang sequences. Comparison of affinities of these concatemers to purified SP120, using a filter binding assay or southwestern blotting, gave an unequivocal result in that only RC showed a significant binding. The binding was abolished effectively by poly(dA). poly(dT) but not by poly(dG).poly(dC), suggesting that the AT-tracts in the RC are the recognition signal for protein binding. It should be emphasized that all the concatemers have identical AT content and the same number of AT-tracts. Thus, the result strongly suggests that the binding activity is accounted for by only a subset of RC molecules with a particular pattern of AT-tracts distribution that are contained in the heterogeneous population. The possible involvement of AT-tracts in the protein recognition was tested more directly with RCs possessing altered AT-tracts. When both AT-tracts were disrupted by replacing T's with G/C or A, all the mutant concatemers showed a marginal binding to SP120, indicating that the intactness of AT-tracts are indeed required for the binding.

  • Research Products

    (12 results)

All Other

All Publications (12 results)

  • [Publications] Kuriyama, M.: "Induction of resistance to etoposide and adriamycin in human glioma cell line treated with antisense oligodeoxynucleotide complementary to the messenger ribonucleic and of deoxyribonucleic acid topoisomerase II alpha"Neurol. Med. Chir.. 37. 655-661 (1997)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Tsutusi, K.: "cDNA cloning of a novel amphiphysin isoform and tissue specific expression of its multiple splice variants"Biochem. Biophys. Res. Commun.. 236. 178-183 (1997)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Tsutusi, K.: "Synthetic Concutemers as artificial MAR : importance of particular configuration of short AT-tracts for protein recognition"Gene Ther. Mol. Biol.. 1. 581-590 (1998)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Akasaka, K.: "Upstream element of the sea urchin arylsulfatase gene serves as an insulator"Cell. Mol. Biol.. 45. 555-565 (1999)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Yabuki, M.: "Caspase activation and cytochrome c release during HL-60 cell apoptosis induced by a nitric oxide donor"Free Radical Res.. (印刷中).

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Maeda, Y.: "Regional differences in the expression of DNA topoisomerase IIβ in the pyramidal neurons of the rat hippocampus"Neurosci. Res.. (印刷中).

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Kuriyama, M.: "Induction of resistance to etoposide and adriamycin in a human glioma cell line treated with antisense oligodexynucleotide complementary to the messenger ribonucleic acid of deoxyribonucleic acid topoisomerase II alpha"Neurol. Med. Chir.. 37. 655-662 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Tsutsui, K.: "cDNA cloning of a novel amphiphysin isoform and tissue-specific expression of its multiple splice variants"Biochem. Biophys. Res. Commun.. 236. 178-183 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Tsustui, K.: "Synthetic concatemers as artificial MAR : importance of a particular configuration of short AT-tract for protein recognition"Gene Ther. Mol. Biol.. 1. 581-590 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Akasaka, K.: "Upstream element of the sea urchin arylsulfatase gene serves as an insulator"Cell. Mol. Biol.. 45. 555-565 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Yabuki, M.: "Caspase activation and cytochrome c release during HL-60 cell apoptosis induced by a nitric oxide donor"Free Radical Res.. (in press).

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Maeda, Y.: "Regional differences in the expression of DNA topoisomerase IIβ in the pyramidal neurons of the rat hippocampus"Neurosci. Res.. (in press).

    • Description
      「研究成果報告書概要(欧文)」より

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Published: 2001-10-23  

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