Sequence specific binding protein of single-stranded and unimolecular quadruplex telomeric DNA from rat hepatocytes

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(C) 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(TAAGGG)(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.