TRF1 binding protein, methods of use thereof

Details TRF1 binding protein, methods of use thereof TRF1 binding protein, methods of use thereof

1998-11-19

2001-08-21

Patterson, Jr., Charles L. (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Transferase other than ribonuclease

Reexamination Certificate

active

06277613

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates generally to a unique vertebrate protein, tankyrase that binds to telomeric repeat binding factor 1 (TRF1), to the nucleic acids encoding tankyrases, and to therapeutic methods of use thereof. The tankyrases may also have a particular use in developing drugs that can counteract the telomere shortening associated with aging and certain diseases such as ataxia telangiectasia.
BACKGROUND OF THE INVENTION
Telomeres are terminal structural elements found at the end of chromosomes [Muller,
The Collecting Net
-
Woods Hole,
13:181-195 (1939)] that protect natural double-stranded DNA ends from degradation, fusion, and recombination with chromosome-internal DNA [McClintock,
Genetics,
26:234-282 (1941); Lundblad et al.,
Cell,
87:369-375 (1996)]. Telomeres are also thought to play a role in the architecture of the nucleus [Agard et al.,
Nature,
302:676-681 (1983); Rabl,
Morphol. J.,
10:214-330 (1885)], and to provide a solution to the end-replication problem that arises as a consequence of successive replication of linear DNA by DNA polymerases which would otherwise result with progressively shorter terminal sequences [Watson,
Nature,
239:197-201 (1972)]. In tetrahymena, impaired telomere function leads to a defect in cytokinesis and to cell death [Yu et al.,
Nature,
344:126-132 (1990)]. Similarly, in yeast, loss of a single telomere results in cell cycle arrest and chromosome instability [Sandell and Zakian,
Cell,
75:729-741 (1993)] and cells undergoing generalized telomere shortening eventually senesce [Lundblad and Szostak,
Cell,
57:633-643 (1989); Singer and Gottschling,
Science,
266:404-409 (1994)].
A ribonucleoprotein reverse transcriptase, telomerase, can elongate telomeres using an internal RNA component as template for the addition of the appropriate G-rich sequence to the 3′ telomere termini [Greider and Blackburn,
Cell,
43:405-413 (1985)]. This activity is thought to compensate for the inability of polymerases to replicate chromosome ends, but other mechanisms of telomere maintenance may operate as well [Pluta et al.,
Nature,
337:429-433 (1989)].
Telomeres contain a tandem array of repeat sequences, typically five to eight base pairs long, that are G-rich in the strand that extends to the end of the chromosome DNA. These repeat units appear to be both necessary and sufficient for telomere function [Lundblad and Szostak,
Cell,
57:633-643 (1989); Szostak et al.,
Cell,
36:459-568 (1982)]. All telomeres of a single genome are composed of the same repeats and these sequences are highly conserved across species. For instance, Oxytricha chromosomes terminate in TTTTGGGG repeats [Klobutcher et al.,
Proc. Natl. Acad. Sci. USA,
78:3015-3019 (1981)], Tetrahymena utilizes an array of (TTGGGG)
n
[Blackburn et al.,
J. Mol. Biol.,
120:33-53 (1978)], and plant chromosomes carry the sequence (TTTAGGG)
n
[Richards et al.,
Cell,
53:127-136 (1988)]. Telomeres of trypanosomes and all vertebrates, including mammals, contain the repeat sequence TTAGGG [Blackburn et al.,
Cell,
36:447-458 (1984); Brown,
Nature,
338:774-776 (1986); Cross et al.,
Nature,
338:771-774 (1989); Moyzis et al.,
Proc. Natl. Acad. Sci. USA,
85:6622-6626 (1988); Van der Ploeg et al.,
Cell,
36:459-468 (1984)]. This 6 basepair sequence is repeated in long tandem arrays at the chromosome ends, which may be as long as 100 kb in the mouse, and varies from 2 to 30 kb in humans [de Lange, Telomere Dynamics and Genome Instability in Human Cancer, In Telomeres, Blackburn and Greider eds., Cold Spring Harbor Press; 265-295 (1995)].
During the development of human somatic tissue, telomeres undergo progressive shortening; in contrast, sperm telomeres increase with donor age [Broccoli et al.,
Proc. Natl. Acad. Sci. USA,
92:9082-9086 (1995); de Lange,
Proc. Natl. Acad. Sci. USA,
91:2882-2885 (1994)]. Most if not all human somatic tissue chromosomes lose terminal TTAGGG repeats with each division, e.g., about 15-40 basepairs per year in the skin and blood. It is unclear what effect this diminution has since human telomeres are between 6-10 kb at birth. On the other hand, it is not yet known how many kilobases of TTAGGG repeats are necessary for optimal telomere function.
Primary human fibroblasts grown in culture lose about 50 basepairs of telomeric DNA per doubling (PD) before they stop dividing at a senescence stage [Allsopp et al.,
Proc. Natl. Acad. Sci. USA,
89:10114-10118 (1992)]. Importantly, there is an excellent correlation between the number of divisions that the cells go through and their initial telomere length. Indeed, it has been suggested that the correlation represents a molecular clock, which limits the potential of primary cells to replicate [Harley et al.,
Nature
(London), 345:458-460 (1990); Harley et al.,
Exp. Gerontol,
27:375-382 (1992)]. Thus, immortalization of human somatic cells involves a mechanism to halt telomere shortening [Bodnar et al.,
Science,
279:349-352 (1998)].
Changes in telomeric dynamics also appear to play a role in the malignant transformation of human cells [Counter et al.,
EMBO J.,
11:1921-1929 (1992); Counter et al.,
Proc. Natl. Acad. Sci. USA,
91:2900-2904 (1994); Kim et al.,
Science,
266:2011-2015 (1994)]. For example, telomeres of tumor cells are generally significantly shorter than those of the corresponding normal cells [de Lange et al.,
Mol. Cell Biol.,
10:518-527 (1990)]. Telomerase activation appears to be an obligatory step in the immortalization of human cells [de Lange,
Proc. Natl. Acad. Sci. USA,
91:2882-2885 (1994); Counter et al.,
EMBO J.,
11:1921-1929 (1992); Counter et al.,
Proc. Natl. Acad. Sci.,
91:2900-2904 (1994); Kim et al.,
Science,
266:2011-2015 (1994); Bodnar et al.,
Science,
279:349-352 (1998)].
Hanish et al. [
Proc. Natl. Acad. Sci. USA,
91:8861-8865 (1994)] examined the requirements for the formation of human telomeres from TTAGGG seeds, and found that telomere formation was not correlated with the ability of human telomerase to elongate telomeric sequences in vitro, and did not appear to be a result of homologous recombination. Rather, the sequence dependence of telomere formation matched the in vitro binding requirements for TRF1, a telomeric TTAGGG repeat binding protein that is associated with human and mouse telomeres in interphase and in mitosis.
Indeed, several observations suggest the existence of regulatory mechanisms to control telomere length. Mammalian telomeres show a species-specific length setting [Kipling and Cooke,
Nature,
347:400-402 (1990)] indicating a mechanism to control telomere length in the germline. Mammalian cells also have a mechanism to measure and regulate the length of individual telomeres. For example, in telomere seed experiments the final length of individual newly-formed telomeres matches the length of the host cell telomeres [Barnett et al.,
Nucl. Acids Res.,
21:27-36 (1993); Hanish et al.,
Proc. Natl. Acad. Sci. USA,
91:8861-8865 (1994)]. Telomere length regulation is also apparent in several human cell lines, which maintain their telomeres at a stable length setting despite high levels of telomerase [Counter et al.,
EMBO J.,
11:1921-1929 (1992)]. Thus, cells can monitor and modulate individual telomeres, a process that is likely to involve proteins bound to the TTAGGG repeats at chromosome ends.
Another process likely to be mediated by TTAGGG binding proteins is the protective cap function of telomeres. Telomeres are protected from the cellular surveillance systems that monitor DNA damage. Thus, cells can distinguish natural chromosome ends (telomeres) from double strand breaks (resulting from DNA damage).
The only known protein components of mammalian telomeres are the TRF proteins, duplex TTAGGG repeat binding factors that are localized a

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