Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
2001-03-26
2004-06-22
Myers, Carla J. (Department: 1634)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C435S193000, C536S023100, C536S023500
Reexamination Certificate
active
06753411
ABSTRACT:
BACKGROUND OF THE INVENTION
Telomeres are the protein-DNA complexes that protect the ends of linear eukaryotic chromosomes from degradation, prevent end-to-end fusions and partake in chromosome localization and segregation (Cooper,
Curr Opin Genet Dev
10: 169-77, 2000; McEachern et al.,
Annu Rev Genet
34: 331-358, 2000; Price,
Curr Opin Genet Dev
9: 218-24, 1999). Telomere length, 15-20 kb in human embryonic or germ line cells, is maintained in part by the enzyme telomerase. In the absence of telomerase activity, about 50-200 bases of DNA are not replicated with each round of cell division, resulting in the eventual diminution in telomere size to typically 5-7 kb. At that length, cells enter a state of arrested growth called replicative senescence. The maintenance of telomere length thus is believed to play a key role in the ability of cells to avoid replicative senescence and to propagate indefinitely, as is the case with stem cells. Likewise, aberrant maintenance of telomere length is believed to underlie indefinite cellular proliferation characteristic of cancer cells (Bodnar et al.,
Science
279: 349-352, 1998; Bryan et al., 1997; McEachern et al., 2000).
Telomeres consist of repeating units of GC-rich DNA and terminate in a single stranded extension of the 3′ strand.
Oxytricha nova
telomeres, for example, consist of tandem repeats of (TTTTGGGG) and end with a 16 nucleotide overhang of the G-rich strand. By contrast, human telomeres have a repeating sequence (TTAGGG)n and end with a 50-100 nucleotide overhang of the G-rich strand. McEachern et al., 2000.
A number of proteins have been identified that specifically interact with the double-stranded portion of the telomere or the single-stranded 3′ extension at its very end. Among the most well characterized are the telomere end-binding proteins from hypotrichous ciliated protozoa (Gottschling et al.,
Cell
47: 195-205, 1986; Price et al.,
Genes Dev
1: 783-93, 1987). The &agr; and &bgr; subunit of the
O. nova
Telomere End-Binding Protein (TEBP) bind specifically to the 16 nucleotide single-stranded extension at the ends of macronuclear chromosomes (Gray et al.,
Cell
67: 807-14, 1991) and form a ternary complex whose structure has been determined using X-ray crystallography (Horvath et al.,
Cell
95: 963-974, 1998). Although both protein subunits directly interact with DNA in the ternary complex, only &agr; binds telomeric DNA by itself (Fang et al.,
Genes Dev
7: 870-82, 1993). The DNA binding domain in the a subunit has been mapped to the N-terminal two-thirds of the polypeptide (Fang et al., 1993) and is comprised of two “OB folds” (Horvath et al., 1998). In vitro reconstituted &agr;-DNA complexes are substrates for telomerase, whereas &agr;-&bgr;-DNA complexes are not; an observation which may indicate a function in the regulation of telomere length (Froelich-Ammon et al.,
Genes Dev
12: 1504-14, 1998).
The protrusion of the G-rich strand as a single-stranded overhang is conserved between ciliates (Klobutcher et al.,
Proc Natl Acad Sci USA
78: 3015-19, 1981), yeast (Wellinger et al.,
Cell
72: 51-60, 1993) and mammalian cells (Makarov et al.,
Cell
88: 657-66, 1997; McElligott et al.,
Embo J
16: 3705-14, 1997; Wright et al.,
Genes Dev
11: 2801-09, 1997), suggesting the existence of similar functional mechanisms in telomere maintenance. However, proteins sharing sequence homology with ciliate TEBPs were not identified in the complete
S. cerevisiae
genome or among the proteins that bind single-stranded telomeric DNA in vitro. Similarly, the
S. cerevisiae
single-stranded telomeric DNA-binding protein cdc13p has not been proposed to be homologous to the ciliate TEBPs, nor have cdc13p homologues been identified in distantly related species. (Ishikawa et al.,
Mol Cell Biol
13: 4301-10, 1993; Lin et al.,
Proc Natl Acad Sci USA
93: 13760-65, 1996; McKay et al.,
Nucleic Acids Res
20: 6461-64, 1992; Nugent et al.,
Science
274: 249-52, 1996; Virta-Pearlman et al.,
Genes Dev
10: 3094-104, 1996).
The apparent absence of specific end-capping proteins in some eukaryotes has been explained by the adoption of a telomere structure distinct from that found in the macronuclei of hypotrichous ciliates. This telomere structure, found at the ends of mammalian and
O. fallax
chromosomes, is a large duplex loop, or “t loop,” created by the sequestration of the single-strand overhang within the double-stranded portion of the telomeric tract (Griffith et al.,
Cell
97: 503-14, 1999; Murti et al.,
Proc Natl Acad Sci USA
96: 14436-39, 1999). In mammals, this architecture is believed to be maintained by a number of proteins, including the TTAGGG-binding factors, TRF1 and TRF2. TRF2 is believed to catalyze the sequestration of the single-stranded DNA into the duplex region of the DNA. Consistent with this notion is the observation that TRF2 can cause telomeric DNA to form t loops in vitro (Griffith et al., 1999). Other proteins have been implicated in telomere architecture and regulation, including TIN2, which was identified by its ability to interact with TRF1 (Kim et al., 1999).
The ability to manipulate telomere structure and metabolism depends on the identification of those components required for the regulation of telomere structure. Evidence has accumulated that telomerase activity itself is not determinative of telomere elongation or replication. For example, some cancer cell lines maintain telomeres in the absence of telomerase activity (Bryan et al., 1997). There is thus a pressing need in the art to identify the functional components that regulate telomere metabolism, to identify compounds that can be used to control the entry, avoidance, or exit of a cell from a state of replicative senescence. Such compounds may be useful alternatively in allowing the indefinite propagation of useful cell lines or in halting the growth of cancer cells in vivo for therapeutic purposes.
SUMMARY OF THE INVENTION
The present invention addresses this need by providing a protein that caps the very ends of human chromosomes, and a related protein that caps the ends of chromosomes in fission yeast (
Schizosaccharomyces pombe
). The protein of the invention is termed “Protection of Telomere-1,” or “Pot1p,” or “Pot1 protein.” Specific embodiments of these proteins are those isolated from humans and fission yeast, hpot1p and SpPot1p, respectively. Polynucleotides encoding a Pot1 protein are also provided.
The inventors have found that Pot1p binds single-stranded telomeric DNA, which is a unforeseen finding, given the apparent absence of end-capping proteins in some eukaryotes. Pot1p both stabilizes chromosome ends and regulates telomerase activity. Accordingly, compounds that stabilize or disrupt the Pot1p-DNA interaction will be useful in regulating the telomere length of a target cell or cell population. The invention thus provides a means of altering cellular life-span, for the purpose of either prolonging the life-span of useful cell populations or making cancer cells enter replicative quiescence. Useful compounds with these properties can be identified through screening methods made possible by the discovery that a Pot1 protein binds single-stranded telomeric DNA. The identification of a Pot1 protein and its encoding DNA also provides a means of developing tools to diagnose illnesses such as cancer that may involve altered expression or structure of a Pot1 protein or gene. Such tools include polynucleotide hybridization probes and antibodies specific for a Pot1 protein.
Accordingly, the invention provides isolated Pot1 proteins having the sequence set forth in SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:9, or SEQ ID NO: 11. Variants of these proteins are capable of binding single-stranded telomeric DNA and have at least 85% sequence identity with, or differ by no more than about 20 single amino acid substitutions, deletions or insertions from, a sequence set forth in SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:9, or SEQ ID NO:11. The invention also provides an isolated, naturally occurring,
Baumann Peter
Cech Thomas R.
Foley & Lardner LLP
Myers Carla J.
The Regents of the University of Colorado
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