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 th e double-stranded region or the guanosine-rich strand and consequently litt le 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 h uman cell extracts that specifically bind the C-rich strand of vertebrate t elomeres [namely a d(CCCTAA)(n) repeat] with high affinity and bind double- stranded telomeric DNA with a 100x reduced affinity. A biochemical assay al lowed us to characterise four proteins of apparent molecular weights 66-64, 45 and 35 kDa, respectively. To identify these polypeptides we screened a lambda gt11-based cDNA expression library, obtained from human HeLa cells u sing a radiolabelled telomeric oligonucleotide as a probe. Two clones were purified and sequenced: the first corresponded to the hnRNP K protein and t he second to the ASF/SF2 splicing factor. Confirmation of the screening res ults was obtained with recombinant proteins, both of which bind to the huma n telomeric C-rich strand in vitro.