Nucleic Acids Research, 2000, Vol. 28, No. 7 1564-1575
© 2000 Oxford University Press
Identification of two human nuclear proteins that recognise the cytosine-rich strand of human telomeres in vitro
Laboratoire de Biophysique, Muséum National d’Histoire Naturelle, INSERM U 201, CNRS UMR 8646, 43 rue Cuvier, 75005 Paris, France, 1IGM, CNRS UMR 5535, 1919 route de Mende, 34293 Montpellier, France, 2INSERM U365, Institut Curie, Recherche Section de Biologie, 26 rue d’Ulm, 75231 Paris cedex 05, France and 3Department of Growth and Reproduction, Section GR-5064, Rigshospitalet, Blegdamsvej 9, DK-2100 Copenhagen, Denmark
Most studies on the structure of DNA in telomeres have been dedicated to the double-stranded region or the guanosine-rich strand and consequently little is known about the factors that may bind to the telomere cytosine-rich (C-rich) strand. This led us to investigate whether proteins exist that can recognise C-rich sequences. We have isolated several nuclear factors from human cell extracts that specifically bind the C-rich strand of vertebrate telomeres [namely a d(CCCTAA)n repeat] with high affinity and bind double-stranded telomeric DNA with a 100x reduced affinity. A biochemical assay allowed us to characterise four proteins of apparent molecular weights 66–64, 45 and 35 kDa, respectively. To identify these polypeptides we screened a 
gt11-based cDNA expression library, obtained from human HeLa cells using a radiolabelled telomeric oligonucleotide as a probe. Two clones were purified and sequenced: the first corresponded to the hnRNP K protein and the second to the ASF/SF2 splicing factor. Confirmation of the screening results was obtained with recombinant proteins, both of which bind to the human telomeric C-rich strand in vitro.
* To whom correspondence should be addressed. Tel: +33 1 40 79 36 89; Fax: +33 1 40 79 37 05; Email: mergny@lovelace.infobiogen.fr Present address: Jérôme Lacoste, Plasticité et Expression des Génomes Microbiens, CNRS EP2029 CEA LRC 12, CERMO, Université Joseph Fourier–Grenoble 1, 460 rue de la Piscine, BP 53, 38041 Grenoble cedex 9, France
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