DOI: 10.1126/science.1060036. Pot1, the Putative Telomere End-Binding Protein in Fission Yeast and Humans

Pot1, the Putative Telomere End-Binding Protein in Fission Yeast and Humans

10.1126/science.1060036

Crossref journal-article

American Association for the Advancement of Science (AAAS)

Science (221)

Abstract

Telomere proteins from ciliated protozoa bind to the single-stranded G-rich DNA extensions at the ends of macronuclear chromosomes. We have now identified homologous proteins in fission yeast and in humans. These Pot1 ( p rotection o f t elomeres) proteins each bind the G-rich strand of their own telomeric repeat sequence, consistent with a direct role in protecting chromosome ends. Deletion of the fission yeast pot1 + gene has an immediate effect on chromosome stability, causing rapid loss of telomeric DNA and chromosome circularization. It now appears that the protein that caps the ends of chromosomes is widely dispersed throughout the eukaryotic kingdom.

Bibliography

Baumann, P., & Cech, T. R. (2001). Pot1, the Putative Telomere End-Binding Protein in Fission Yeast and Humans. Science, 292(5519), 1171â1175.

Authors 2

Peter Baumann (first)
Thomas R. Cech (additional)

References 42 Referenced 815

10.1073/pnas.78.5.3015
10.1016/0092-8674(93)90049-V
The presence of 3′ single-stranded extensions in S. pombe was verified by hybridization of native genomic DNA with a telomeric probe before and after exonuclease I treatment (25).
10.1016/S0092-8674(00)81908-X
10.1093/emboj/16.12.3705
10.1101/gad.11.21.2801
10.1128/MCB.15.11.6128
10.1016/S0092-8674(01)00226-4
10.1126/science.286.5437.117
10.1016/0092-8674(86)90442-3
10.1016/0092-8674(91)90075-A
The O. nova α subunit by itself preferentially binds the 3′ terminus of a telomeric oligonucleotide but also binds single-stranded telomeric DNA internally (11).
10.1073/pnas.90.13.6056
10.1016/S0092-8674(00)81720-1
10.1093/nar/20.24.6621
10.1016/S0092-8674(00)80760-6
Heterozygous diploids (h + /h – leu1-32/leu1-32 ura4-D18 / ura4-D18 his3-D1 / his3-D1 ade6-M210 / ade6-M216 pot1 + / pot1::kan r ) were constructed by replacing the entire ORF of pot1 + with the kanamycin resistance gene as described (34).
10.1126/science.282.5388.493
P. Baumann T. R. Cech data not shown.
10.1038/2517
The S. pombe pot1 + ORF plus an NH 2 -teminal His 6 tag and COOH-terminal V5/His 6 tag was expressed in E. coli strain M15 (pRep4) using tryptone phosphate media. After induction (0.8 mM isopropyl-β-D-thiogalactopyranoside) for 6 hours at 24°C cells were harvested and resuspended in 50 mM NaH 2 PO 4 (pH 8.0) 0.1 M NaCl 2 mM imidazole 10% glycerol 0.2% Tween-20 5 mM β-mercaptoethanol and 1 mM phenylmethylsulfonyl fluoride to which lysozyme (0.5 mg/ml) was then added. After 30 min the concentration of NaCl was increased to 0.6 M genomic DNA was sheared by sonication and cell debris was removed by centrifugation. The supernatant was incubated at 4°C for 90 min with Ni-NTA resin (Qiagen) which was then loaded onto a column and washed sequentially with P buffer [50 mM NaH 2 PO 4 (pH 8.0) 0.6 M NaCl 10% glycerol 0.2% Tween-20 and 5 mM β-mercaptoethanol] containing increasing concentrations of imidazole. Pot1p eluted around 90 mM imidazole. Pot1p-containing fractions were dialyzed against T buffer [50 mM tris-HCl (pH 8.0) 10% glycerol 0.5 mM EDTA and 0.5 mM dithiothreitol] containing 0.2 M KCl and Pot1p was further purified on a Q-sepharose column (Pharmacia) using a linear gradient of KCl (0.2 to 1 M). The 22-kD Pot1p fragment was found in the flowthrough whereas Pot1p eluted around 0.5 M KCl. The protein was dialyzed against T buffer plus 0.2 M KCl and stored at –80°C.
Identical results were obtained when hemagglutinin-tagged Pot1p was expressed in S. pombe and cell-free extracts were used in bandshift assays (19).
10.1101/gad.7.5.870
10.1126/science.7569926
See Science Online (www.sciencemag.org/cgi/content/full/292/5519/1171/DC1).
10.1126/science.277.5328.955
10.1016/S0092-8674(00)80538-3
10.1093/hmg/6.12.2011
Human POT1 was amplified by polymerase chain reaction from ovary cDNA and cloned into the pQE30 expression vector. The protein was purified over Ni-NTA resin under the same conditions as SpPot1p. The human protein eluted around 135 mM imidazole.
10.1101/gad.12.10.1504
10.1002/j.1460-2075.1993.tb05726.x
Yet another state may involve a complex with heterogeneous nuclear ribonucleoproteins several of which have properties consistent with a role in telomere maintenance (35–38).
Pot1p might also bind the displaced single strand of a D-loop within a t-loop although the protein does have a preference for binding terminal rather than internal repeats.
10.1091/mbc.11.10.3265
Ishikawa F., Matunis M. J., Dreyfuss G., Cech T. R., Mol. Cell. Biol. 13, 4301 (1993). / Mol. Cell. Biol. by Ishikawa F. (1993)
10.1093/nar/20.24.6461
10.1038/575
10.1128/MCB.20.15.5425-5432.2000
10.1093/nar/19.20.5515
10.1007/s004120050311
10.1093/nar/17.7.2801
We thank D. Lyons G. Mellitzer T. Nakamura O. Peersen V. Wood and the members of the Cech laboratory for helpful discussions; D. King for mass spectroscopy; D. Baumann K. Goodrich Y. Han and E. Podell for technical assistance; and T. Bryan K. Friedman and A. Zaug for critical reading of the manuscript. P.B. was supported in part by a Wellcome Prize Traveling Research Fellowship (grant 054549/Z/98/Z).

Dates

Type	When
Created	23 years, 1 month ago (July 27, 2002, 5:52 a.m.)
Deposited	1 year, 7 months ago (Jan. 9, 2024, 5:31 p.m.)
Indexed	1 month, 1 week ago (July 26, 2025, 5:34 a.m.)
Issued	24 years, 3 months ago (May 11, 2001)
Published	24 years, 3 months ago (May 11, 2001)
Published Print	24 years, 3 months ago (May 11, 2001)

Funders 0

None

BibTeX

@article{Baumann_2001, title={Pot1, the Putative Telomere End-Binding Protein in Fission Yeast and Humans}, volume={292}, ISSN={1095-9203}, url={http://dx.doi.org/10.1126/science.1060036}, DOI={10.1126/science.1060036}, number={5519}, journal={Science}, publisher={American Association for the Advancement of Science (AAAS)}, author={Baumann, Peter and Cech, Thomas R.}, year={2001}, month=may, pages={1171–1175} }

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  "abstract": "<jats:p>\n            Telomere proteins from ciliated protozoa bind to the single-stranded G-rich DNA extensions at the ends of macronuclear chromosomes. We have now identified homologous proteins in fission yeast and in humans. These Pot1 (\n            <jats:underline>p</jats:underline>\n            rotection\n            <jats:underline>o</jats:underline>\n            f\n            <jats:underline>t</jats:underline>\n            elomeres) proteins each bind the G-rich strand of their own telomeric repeat sequence, consistent with a direct role in protecting chromosome ends. Deletion of the fission yeast\n            <jats:italic>pot1</jats:italic>\n            <jats:sup>+</jats:sup>\n            gene has an immediate effect on chromosome stability, causing rapid loss of telomeric DNA and chromosome circularization. It now appears that the protein that caps the ends of chromosomes is widely dispersed throughout the eukaryotic kingdom.\n          </jats:p>",
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