Abstract
The Pot1 (protection of telomeres) protein binds to single-stranded telomeric DNA and is essential for the protection of chromosome ends from degradation and end-to-end fusions. The Pot1 amino-terminal DNA binding domain, Pot1N, adopts an oligonucleotide/oligosaccharide binding fold and binds GGTTAC motifs cooperatively and with exceptionally high sequence specificity. We have now examined DNA binding to naturally occurring telomeric substrates based on the analysis of 100 cloned chromosome ends and in the context of the full-length Pot1 protein. Here, we describe several important differences between Pot1 and Pot1N with apparent consequences for chromosome end protection. Specifically, full-length Pot1.DNA complexes are more stable, and the minimal binding site for a Pot1 monomer is extended into two adjacent telomeric repeats. We provide evidence that Pot1 contains a second DNA binding motif that recognizes DNA with reduced sequence specificity compared with the domain present in Pot1N. The two DNA binding motifs cooperate, whereby the amino-terminal oligonucleotide/oligosaccharide binding fold determines the registry of binding, and the internal DNA binding motif stabilizes the complex and expands the protected region toward the 3' -end. Consistent with a role in chromosome end capping, Pot1 prevents access of telomerase to the 3'-end and protects against exonucleolytic degradation.
Highlights
Sequence-specific, single-stranded DNA-binding proteins include telomere end-binding proteins (TEBPs)1 from ciliated protozoa (9 –11), Cdc13 from the budding yeast [12, 13], and the protection of telomeres (Pot1) family of proteins found in fungi, plants, and vertebrates [14, 15]
Expression and Purification of Recombinant Pot1 Protein— Pot1 and Pot1N were expressed in E. coli, and soluble extracts of several clones were analyzed for expression by Western blotting
The fission yeast Pot1 protein is critical for chromosome end protection, as evidenced by the rapid loss of telomeres when Pot1 function is lost
Summary
Sequence-specific, single-stranded DNA-binding proteins include telomere end-binding proteins (TEBPs) from ciliated protozoa (9 –11), Cdc from the budding yeast [12, 13], and the protection of telomeres (Pot1) family of proteins found in fungi, plants, and vertebrates [14, 15]. Cdc shares no apparent sequence similarity with the ciliate proteins, the NMR structure of the Cdc DNA binding domain revealed the presence of an OB-fold that is structurally similar to the amino-terminal OB-fold of the TEBP ␣ subunit [26]. Initial characterization of fission yeast, human, and chicken Pot demonstrated that they bind to the cognate G strand of telomeric DNA and play a critical role in telomere length regulation and end protection [14, 27,28,29]. Pot Binding to Natural Telomeres has been mapped to include the 185 amino-terminal residues of Pot1 This fragment, here referred to as Pot1N, retains the binding specificity of the full-length protein, and the high resolution crystal structure of Pot1N in complex with DNA has been solved [32]. Any sequence change in the DNA prevents this structure from forming, providing an explanation for the high sequence specificity in DNA binding
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