Purification, characterization and molecular cloning of TGP1, a novel G-DNA binding protein from Tetrahymena thermophila.
G-DNA, a polymorphic family of four-stranded DNA structures, has been proposed to play roles in a variety of biological processes including telomere function, meiotic recombination and gene regulation. Here we report the purification and cloning of TGP1, a G-DNA specific binding protein from Tetrahymena thermophila. TGP1 was purified by three-column chromatographies, including a G-DNA affinity column. Two major proteins (approximately 80 and approximately 40 kDa) were present in the most highly purified column fraction. Renaturation experiments showed that the approximately 80 kDa protein contains TGP1 activity. Biochemical characterization showed that TGP1 is a G-DNA specific binding protein with a preference for parallel G-DNAs. The TGP1/DNA complex has a dissociation constant (Kd) of approximately 2.2 x 10(-8) M and TGP1 can form supershift in gel mobility shift assays. The cDNA coding TGP1 was cloned and sequenced based upon an internal peptide sequence obtained from the approximately 80 kDa protein. Sequence analyses showed that TGP1 is a basic protein with a pI of 10.58, and contains two extensively hydrophilic and basic domains. Homology searches revealed that TGP1 is a novel protein sharing weak similarities with a number of proteins.
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Department of Zoology and Genetics and Molecular, Cellular and Developmental Biology Program, Iowa State University, Ames, IA 50011, USA.
Schierer
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Kang
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Henderson
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D016054:DNA, Protozoan / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D016808:Tetrahymena thermophila / Q000378:metabolism*
D016054:DNA, Protozoan; D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; C112058:TGP1 protein, Tetrahymena