ABSTRACT
We have purified a 100 kDa protein, resolved in a Southwestern binding screen of total nuclear proteins from Hela cells with double-stranded human telomeric probe. A polyclonal antiserum raised by this protein recognizes purified nucleolin and stains nucleoli in growing Hela cells. We demonstrate that a truncated form of human nucleolin and a purified deletion derivative of mouse nucleolin bind in vitro to duplex telomeric DNA. This study suggests a new link between telomeres and the nucleolus.
Subject(s)
DNA/metabolism , Nuclear Proteins/metabolism , Phosphoproteins/metabolism , RNA-Binding Proteins/metabolism , Telomere/metabolism , Animals , Antibodies , Base Sequence , Binding Sites/genetics , Cell Nucleolus/metabolism , DNA/genetics , Escherichia coli/genetics , HeLa Cells , Humans , In Vitro Techniques , Mice , Nuclear Proteins/genetics , Phosphoproteins/genetics , Protein Binding , RNA-Binding Proteins/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , NucleolinABSTRACT
Natural chromosomal ends are stabilized by proteins that bind duplex telomeric DNA repeats. In human cells, the TTAGGG Repeat Factor 1 (TRF1) was identified by two independent studies, one screening for factors that bind duplex telomeric DNA and the other screening for proteins containing a particular Myb motif called the telobox, which is required for telomeric repeat recognition (Fig. 1a; refs 3-5). A second human open reading frame, orf2, contains a telobox sequence and encodes a polypeptide that specifically recognizes mammalian telomeric repeat DNA in vitro. We show that two proteins of 65 and 69 kD, expressed in HeLa cells, contain the orf2 telobox sequence. These proteins are collectively termed TRF2. Affinity-purified antibodies specific for anti-TRF2 label the telomeres of intact human chromosomes, strengthening the correlation between occurrence of telobox and telomere-repeat recognition in vivo.
Subject(s)
DNA-Binding Proteins/metabolism , Telomere/metabolism , Amino Acid Sequence , Base Sequence , DNA/genetics , DNA/metabolism , DNA Primers/genetics , DNA-Binding Proteins/genetics , Fluorescent Antibody Technique , HeLa Cells , Humans , Microsatellite Repeats , Molecular Sequence Data , Open Reading Frames , Polymerase Chain Reaction , Telomere/genetics , Telomeric Repeat Binding Protein 2ABSTRACT
The yeast TTAGGG binding factor 1 (Tbf1) was identified and cloned through its ability to interact with vertebrate telomeric repeats in vitro. We show here that a sequence of 60 amino acids located in its C-terminus is critical for DNA binding. This sequence exhibits homologies with Myb repeats and is conserved among five proteins from plants, two of which are known to bind telomeric-related sequences, and two proteins from human, including the telomeric repeat binding factor (TRF) and the predicted C-terminal polypeptide, called orf2, from a yet unknown protein. We demonstrate that the 111 C-terminal residues of TRF and the 64 orf2 residues are able to bind the human telomeric repeats specifically. We propose to call the particular Myb-related motif found in these proteins the 'telobox'. Antibodies directed against the Tbf1 telobox detect two proteins in nuclear and mitotic chromosome extracts from human cell lines. Moreover, both proteins bind specifically to telomeric repeats in vitro. TRF is likely to correspond to one of them. Based on their high affinity for the telomeric repeat, we predict that TRF and orf2 play an important role at human telomeres.
