Identification of two human nuclear proteins that recognise the cytosine-rich strand of human telomeres in vitro.
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 100xreduced 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 alambdagt11-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.
Lacroix
L
L
Laboratoire de Biophysique, Muséum National d'Histoire Naturelle, INSERM U 201, CNRS UMR 8646, 43 rue Cuvier, 75005 Paris, France.
Liénard
H
H
Labourier
E
E
Djavaheri-Mergny
M
M
Lacoste
J
J
Leffers
H
H
Tazi
J
J
Hélène
C
C
Mergny
J L
JL
D001482:Base Composition; D001483:Base Sequence; D001665:Binding Sites; D003596:Cytosine / Q000737:chemistry; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D034641:Heterogeneous-Nuclear Ribonucleoprotein K; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D006801:Humans; D008970:Molecular Weight; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000068103:Serine-Arginine Splicing Factors; D016615:Telomere / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D034641:Heterogeneous-Nuclear Ribonucleoprotein K; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D009687:Nuclear Proteins; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins; D000068103:Serine-Arginine Splicing Factors; D003596:Cytosine; D004247:DNA