Purification and characterization of qTBP42, a new single-stranded and quadruplex telomeric DNA-binding protein from rat hepatocytes

Abstract

Telomeres of vertebrate chromosomes terminate with a short 5'-d(TTAGGG)- 3' single-stranded overhang that can form in vitro tetrahelical structures. Here we describe a new protein from rat hepatocyte nuclei designated quadruplex telomere-binding protein 42 (qTBP42) that tightly binds 5'- d(TTAGGG)(n)-3' and 5'-d(CCC-TAA)(n)-3' single-stranded and tetraplex forms of 5'd(TTAGGG)(n)-3'. The thermostable qTBP42 was isolated from boiled nuclear extracts and purified to near homogeneity by successive steps of column chromatography on DEAE-cellulose, phosphocellulose, and phenyl- Sepharose. A subunit molecular size of 42.0 ± 2.0 kDa was determined for qTBP42 by Southwestern blotting and SDS-polyacrylamide gel electrophoresis of the protein and its UV cross-linked complex with labeled telomeric DNA. A native size of 53.5 ± 0.9 kDa, estimated by Superdex 200 gel filtration, suggests that qTBP42 is a monomeric protein. Sequences of five tryptic peptides of qTBP42 contained motifs shared by a mammalian CArG box-binding protein, hnRNP A/B, hnRNP C, and a human single-stranded telomeric DNA- binding protein. Complexes of qTBP42 with each complementary strand of telomeric DNA and with quadruplex forms of the guanine-rich strand had 3.7- 14.6 nM dissociation constants, K(d), whereas complexes with double-stranded telomeric DNA had up to 100-fold higher K(d) values. By associating with tetraplex and single-stranded telomerie DNA, qTBP42 increased their heat stability and resistance to digestion by micrococcal nuclease.