Purification and characterization of qTBP42, a new single-stranded and quadruplex telomeric DNA-binding protein from rat hepatocytes.
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(CCCTAA)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 copyright 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, Kd, whereas complexes with double-stranded telomeric DNA had up to 100-fold higher Kd values. By associating with tetraplex and single-stranded telomeric DNA, qTBP42 increased their heat stability and resistance to digestion by micrococcal nuclease.
Sarig
G
G
Unit of Biochemistry, the Bruce Rappaport Faculty of Medicine, Technion, Israel Institute of Technology, P.O. Box 9649, Haifa 31096 Israel.
Weisman-Shomer
P
P
Erlitzki
R
R
Fry
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D015139:Blotting, Southern; D015153:Blotting, Western; D002848:Chromatography, DEAE-Cellulose; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000302:isolation & purification* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D008969:Molecular Sequence Data; D016601:RNA-Binding Proteins / Q000378:metabolism; D051381:Rats; D016615:Telomere; D034501:Telomere-Binding Proteins
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D016601:RNA-Binding Proteins; D034501:Telomere-Binding Proteins; C440851:quadruplex telomeric DNA-binding protein 42, rat