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Volume 272, Number 25, Issue of June 20, 1997 pp. 15881-15890
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Sequence-specific Binding Protein of Single-stranded and Unimolecular Quadruplex Telomeric DNA from Rat Hepatocytes

(Received for publication, February 10, 1997, and in revised form, April 16, 1997)

From the Unit of Biochemistry, The Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, P. O. Box 9649, Haifa 31096, Israel

A rat liver nuclear protein, unimolecular quadruplex telomere-binding protein 25, (uqTBP25) is described that binds tightly and specifically single-stranded and unimolecular tetraplex forms of the vertebrate telomeric DNA sequence 5-d(TTAGGG)_n-3. A near homogeneous uqTBP25 was purified by ammonium sulfate precipitation, chromatographic separation from other DNA binding proteins, and three steps of column chromatography. SDS-polyacrylamide gel electrophoresis and Superdex© 200 gel filtration disclosed for uqTBP25 subunit and native M_r values of 25.4 ± 0.5 and 25.0 kDa, respectively. Sequences of uqTBP25 tryptic peptides were closely homologous, but not identical, to heterogeneous nuclear ribonucleoprotein A1, heterogeneous nuclear ribonucleoprotein A2/B1, and single-stranded DNA-binding proteins UP1 and HDP-1. Complexes of uqTBP25 with single-stranded or unimolecular quadruplex 5-d(TTAGGG)₄-3, respectively, had dissociation constants, K_d, of 2.2 or 13.4 nM. Relative to d(TTAGGG)₄, complexes with 5-r(UUAGGG)₄-3, blunt-ended duplex telomeric DNA, or quadruplex telomeric DNA had >10 to >250-fold higher K_d values. Single base alterations within the d(TTAGGG) repeat increased the K_d of complexes with uqTBP25 by 9-215-fold. Association with uqTBP25 protected d(TTAGGG)₄ against nuclease digestion, suggesting a potential role for the protein in telomeric DNA transactions.

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