SEQUENCE-SPECIFIC BINDING-PROTEIN OF SINGLE-STRANDED AND UNIMOLECULARQUADRUPLEX 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 te lomeric DNA sequence 5'-d(TTAGGG)(n)-3', A near homogeneous uqTBP25 wa s purified by ammonium sulfate precipitation, chromatographic separati on from other DNA binding proteins, and three steps of column chromato graphy, 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 pep tides were closely homologous, but not identical, to heterogeneous nuc lear 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', blun t ended duplex telomeric DNA, or quadruplex telomeric DNA had >10 to > 250-fold higher K-d values, Single base alterations within the d(TTAGG G) repeat increased the K-d of complexes with uqTBP25 by 9-215-fold, A ssociation with uqTBP25 protected d(TTAGGG)(4) against nuclease digest ion, suggesting a potential role for the protein in telomeric DNA tran sactions.