SUNY at Albany
June 19-23, 2001
Association of yeast inosine 5'-monophosphate dehydrogenase with a fragment of the C-rich telomeric strand
Stretches of cytidine occur in the C-rich strand of telomeric or centromeric DNA. A DNA fragment containing four repeats of the vertebrate telomeric DNA, CCCTAA, forms an intramolecular i-motif structure whose geometry and loop motions have been elucidated in detail by NMR (2). Other cases of i-motif structures have been reported for telomeric and centromeric sequences, and for related DNA sequences (reviewed in (3)). Such observations suggest that the i-motif could exist in vivo. If so, it would presumably interact with one or more proteins..
We therefore decided to search for such a protein, and chose for the i-motif-forming sequence a stretch of the C-rich strand of telomeres of S. cerevisiae, an eucaryote whose genome has been completely sequenced. Each telomere of the sixteen chromosomes includes tandem repeats of C2-3A(CA)1-6 / (TG)1-6TG2-3 over a length of 75 to 300 base pairs (4). We chose the sequence d[(C3ACA)3C3] which is found in chromosomes I, VIII, XII and XIII, and which forms an intra-molecular i-motif in vitro (J-F. Cornuel, Doctoral Thesis, Ecole polytechnique (2000)). Using native gel retardation as the primary assay, we characterized a protein multimer which associates to the DNA sequence with moderate to strong specificity. Gel retardation measurements in the range of pH 6 to 8 showed no effect of pH, indicating that the DNA fragment might form an intramo-lecular i-motif structure in the complex. The proteins were traced by a differential affinity technique and identified by mass spectrometry. They correspond to three closely related genes, IMD2, IMD3 and IMD4, each of which codes for a probable inosine monophosphate dehydrogenase. The genes IMD2 and IMD3 are located close to the telomere, and could therefore be subject to silencing by the telomere position effect. We have also observed retardation of a human telomeric fragment by incubation with HeLa nuclear extract, apparently analogous to that characterized here for the yeast system.References and Footnotes
Jean-Fran?ois Cornuel, Anne Moraillon and Maurice Gu?ron
Groupe de Biophysique de l'UMR 7643 du CNRS, Ecole Polytechnique,
91128 Palaiseau, France.