Protection-of telomere-1 (POT-1) protein and encoding polynucleotides

Abstract

A protein identified in humans and Schizosaccharomyces pombe , Pot1p, binds single-stranded telomeric DNA and both stabilizes chromosome ends and regulates telomerase activity. Compounds that stabilize or disrupt the Pot1p-DNA interaction will be useful in regulating the telomere length of a cell. Because telomere length is involved in the regulation of cellular life-span, the life-span of useful cell populations may be prolonged or undesirable cells may be caused to cease proliferation. The identification of a Pot1 protein and its encoding DNA provides methods of screening useful compounds or diagnosing illnesses that involve altered expression or structure of a Pot1 protein or gene.

What is claimed is:  
     
         1 . A method of identifying a compound that regulates the binding of Pot1 to telomeric DNA, comprising detecting whether a candidate compound regulates the binding of a Pot1 polypeptide to a single-stranded telomeric DNA.  
     
     
         2 . The method of  claim 1 , wherein the candidate compound is exposed to a Pot1 polypeptide-telomeric DNA complex.  
     
     
         3 . The method of  claim 1 , wherein the candidate compound is exposed to the Pot1 polypeptide prior to exposure to the telomeric DNA.  
     
     
         4 . The method of  claim 1 , wherein the step of detecting comprises detecting whether the candidate compound strengthens the interaction between the Pot1 polypeptide and the telomeric DNA.  
     
     
         5 . The method of  claim 1 , wherein the step of detecting comprises detecting whether the candidate compound stabilizes the interaction between the Pot1 polypeptide and the telomeric DNA.  
     
     
         6 . The method of  claim 1 , wherein the step of detecting comprises detecting whether the candidate compound weakens the interaction between the Pot1 polypeptide and the telomeric DNA.  
     
     
         7 . The method of  claim 1 , wherein the step of detecting comprises detecting whether the candidate compound disrupts the interaction between the Pot1 polypeptide and the telomeric DNA.  
     
     
         8 . The method of  claim 1 , wherein the step of detecting comprises detecting whether the candidate compound interacts with the Pot1 polypeptide or a complex between the Pot1 polypeptide and the telomeric DNA to change the binding constant of the complex between the Pot1 polypeptide and the telomeric DNA.  
     
     
         9 . The method of  claim 1 , wherein the step of detecting is performed by detecting the ability of the candidate compound to change the amount of a labeled probe comprising a fragment of single-stranded telomeric DNA that interacts with the Pot1 polypeptide.  
     
     
         10 . The method of  claim 1 , wherein the step of detecting is performed using an electrophoretic mobility shift assay.  
     
     
         11 . The method of  claim 1 , wherein the step of detecting is performed using a high throughput assay for screening candidate compounds simultaneously.  
     
     
         12 . The method of  claim 1 , wherein the step of detecting is performed using an isolated cell that recombinantly expresses the Pot1 polypeptide.  
     
     
         13 . The method of  claim 1 , further comprising testing candidate compounds that regulate the binding of a Pot1 polypeptide to single-stranded telomeric DNA to determine whether the candidate compounds regulate telomere length or integrity throughout repeated divisions in a cell culture system.  
     
     
         14 . The method of  claim 1 , wherein the Pot1 polypeptide is selected from the group consisting of: 
 a) a Pot1 polypeptide comprising an amino acid sequence selected from the group consisting of: SEQ ID NO:5, SEQ ID NO:13, SEQ ID NO:15 and SEQ ID NO:17.    b) a Pot1 polypeptide comprising an amino acid sequence that is at least about 85% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO:5, SEQ ID NO:13, SEQ ID NO:15 and SEQ ID NO:17, wherein the polypeptide binds single-stranded telomeric DNA; and    c) a fragment of a Pot1 polypeptide as set forth in (a) or (b), wherein the fragment binds single-stranded telomeric DNA.    
     
     
         15 . The method of  claim 1 , wherein the Pot1 polypeptide comprises an amino acid sequence that is at least about 90% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO:5, SEQ ID NO:13, SEQ ID NO:15 and SEQ ID NO:17, wherein the polypeptide binds single-stranded telomeric DNA.  
     
     
         16 . The method of  claim 1 , wherein the Pot1 polypeptide is a fragment of an amino acid sequence selected from the group consisting of: SEQ ID NO:5, SEQ ID NO:13, SEQ ID NO:15 and SEQ ID NO:17, wherein the fragment binds single-stranded telomeric DNA.  
     
     
         17 . The method of  claim 1 , wherein the Pot1 polypeptide comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:5, SEQ ID NO:13, SEQ ID NO:15 and SEQ ID NO:17.  
     
     
         18 . The method of  claim 1 , wherein the Pot1 polypeptide comprises SEQ ID NO:5.  
     
     
         19 . The method of  claim 1 , wherein the single-stranded telomeric DNA is G-rich.  
     
     
         20 . The method of  claim 1 , wherein the single-stranded telomeric DNA comprises TTAGGG (positions 1-6 of SEQ ID NO:20) repeats.  
     
     
         21 . The method of  claim 1 , wherein the single-stranded telomeric DNA comprises a nucleic acid sequence selected from the group consisting of any one of SEQ ID NOs:36-38.  
     
     
         22 . The method of  claim 1 , wherein the candidate compound is selected from the group consisting of: a small organic molecule, an oligonucleotide, and a non-hydrolyzable DNA analogue.  
     
     
         23 . A method of identifying a compound that interferes with the binding of a Pot1 polypeptide to a single-stranded telomeric DNA, comprising determining whether the candidate compound decreases the binding of the Pot1 polypeptide to a single-stranded telomeric DNA molecule in a mixture comprising the single-stranded telomeric DNA molecule, the polypeptide, and the candidate compound.