Purification and Characterization of qTBP42, a New Single-stranded and Quadruplex Telomeric DNA-binding Protein from Rat Hepatocytes

Abstract

Telomeres, the DNA-protein structures at the end of linear eukaryotic chromosomes, form protective caps that shield chromosome termini against degradative processes or fusion with other chromosome ends (1-4). The evolutionarily conserved nucleotide sequence of telomeric DNA consists of short nucleotide sequences repeated in tandem. All vertebrates, slime molds, filamentous fungi, and Trypanosoma have a repeated 5Ј-d(TTAGGG)-3Ј sequence of the telomeric strand oriented 5Ј to 3Ј toward the chromosome terminus, the "G-strand". The 3Ј-terminal G-strand stretch ends with an unpaired 12-16-nucleotide-long overhang (1-4). This single-stranded tract can form in vitro under physiological conditions hairpin (5-7) or unimolecular or bimolecular tetrahelical structures (5-13), which may function in telomere transactions.The telomere hypothesis of cellular senescence and tumorigenesis states that progressive shortening of telomeres in somatic cells leads to cessation of their division and to cellular aging (16). In contrast, maintenance of a stable telomere length in germ line and cancer cells is associated with their infinite division and immortality. This hypothesis is supported by evidence showing that whereas telomeric DNA becomes progressively shortened in the course of the aging of diverse somatic cells, its length remains stable in infinitely dividing stem and tumor cells (17-21). The G-strand of telomeric DNA is extended by telomerase, a ribonucleoprotein enzyme whose complementary cytosine-rich RNA component serves as a template for the synthesis of the G-strand (3,4,22). Whereas telomerase activity is undetectable in various somatic tissues and in dividing primary cells, many cancer cells retain an active telomerase reviewed in Ref. 17). More recent results suggest, however, that telomerase might not be the sole factor that determines telomere length. Thus, some tumor cells whose telomere length remains stable have no measurable telomerase activity (24), and conversely, an active telomerase was detected in normal human (25) and mouse cells (26). Further, the removal of 50 -200-nucleotide-long segments of telomeric DNA with each round of replication in somatic cells (27,28) suggests that, in addition to the loss of telomerase activity, termini of telomeric DNA may also be shortened by exonucleolytic attack.Stabilization or destabilization of telomeric DNA may be affected by telomeric DNA-binding proteins that have been identified in diverse species. Proteins such as a monomeric 51-kDa polypeptide from Euplotes crassus (29), a 34-kDa Chlamydomonas protein (30), and a Xenopus protein (31) bind tightly to single-stranded telomeric DNA. Another group of proteins bind to or accelerate the formation of tetraplex structures of telomeric DNA. A heterodimeric protein from Oxytricha nova consisting of a 56-kDa ␣ and a 41-kDa ␤ subunit, dimerizes in the presence of single-stranded telomeric DNA and binds to it cooperatively (32). Without detectably binding to quadruplex DNA, the ␤ subunit facilitates tetraplex formation 10 5 ...

Tetrahelical Forms of the Fragile X Syndrome Expanded Sequence d(CGG) Are Destabilized by Two Heterogeneous Nuclear Ribonucleoprotein-related Telomeric DNA-binding Proteins

“…The hnRNP-related proteins qTBP42 and uqTBP25 tightly bind and stabilize single-stranded and tetraplex telomeric sequences (37,39). However, a survey of the interaction of qTBP42 and uqTBP25 with tetraplex structures of different DNA sequences revealed that neither protein detectably bound d(CGG) n tetrahelices.…”

“…Previously identified as proteins that tightly bind and stabilize quadruplex telomeric DNA (37)(38)(39), it is shown here that qTBP42 and uqTBP25 destabilized bimolecular tetraplex structures of d(CGG) n without detectably binding these tetrahelices. Destabilization of tetraplex d(CGG) n by either protein did not require ATP or Mg 2ϩ or the presence of an unpaired single-strand tail at the ends of the tetrahelix.…”

“…In this paper we report that two hnRNP-related nuclear DNA-binding proteins from rat liver, quadruplex telomeric DNA-binding protein 42 (qTBP42) (37,38), and unimolecular quadruplex telomeric DNA-binding protein 25 (uqTBP25) (39) destabilized tetraplex structures of d(CGG) n . Previously identified as proteins that tightly bind and stabilize quadruplex telomeric DNA (37)(38)(39), it is shown here that qTBP42 and uqTBP25 destabilized bimolecular tetraplex structures of d(CGG) n without detectably binding these tetrahelices.…”

A chicken hnRNP of the A/B family recognizes the single‐stranded d(CCCTAA)n telomeric repeated motif

“…Interestingly, a protein from Trypanosoma, ST-1, binds to the telomeric double-stranded repeat as well as to its singlestranded C-rich component [14]. In vertebrates, the nuclear protein from rat hepatocytes, qTBP42, has been shown to recognize each of the single-stranded forms of the telomeric repeat [9]. Several proteins that interact with polypyrimidine ssDNA have been described.…”

“…Another group of proteins, ST-2 from Trypanosoma [8], qTBP42 from rat [9], human replication factor C [10], and murine STBP [11] and A1/UP1 [12], also bind to the single-stranded G-rich telomeric motif, although their function has not been fully ascertained. The last of these, however, is the first ssDNA-binding protein shown to be directly involved in mammalian telomere biogenesis, suggesting a possible mechanism by which telomere length can be modulated [13].…”