Skip to Main content Skip to Navigation
Theses

Complexation d'un fragment de brin télomérique riche en C par des protéines de Saccharomyces cerevisiae. Identification génomique aux IMPDH. Possible repliement en motif i de l'ADN complexé.

Abstract : The final areas (telomere) of the chromosomes of the majority of the organizations are essential with the maintenance and the integrity at the end of the chromosomes and are implied in the cancerogenesis and cellular ageing. The telomeres are formed by one rich strand in guanines and one strand rich in cytosines. In vitro, each strand is organized in a folded up structure, quadruplexe of guanine for one, and the i-motif for the other, which raises the question of the biological role of these structures. Several proteins bind to the G strand and supporting the formation of guanine quadruplexes are known. Two proteins specifically recognizing the C strand of human telomeric DNA were identified (ASF/SF2 and hnRNP K). We wanted to contribute to the comprehension of the mechanisms of regulation of the telomeres by seeking a protein specifically binding to a sequence telomeric DNA rich in cytosine. This study was undertaken on an organization eucaryote, the yeast Saccharomyces cerevisiae. We started by showing that the fragment telomeric DNA of yeast, d[(CCCACA)3CCC], could form an i-motif in vitro. We observed by nondenaturing electrophoretic migration, the formation one complex DNA-protein on which we focused our efforts. The molecular mass of this protein (~ 250 kDa) was determined by gel filtration chromatography. The association DNA-protein was observed for a range of pH from 6 to 8. One duplexes ADN B d(CGCGATCGCG) or one small continuation of cytosines d(CCC) are not candidates and the delayed signal is attenuated of half for a report/ratio monobrin d(T14)/probe of 4000. The ADN of E coli, crossed in fragments from 10 to 1000 bases, is candidate (50%) for a competitor/probe ratio equal to 1000. But all oligomer sequences tested in vitro formant an i-motif monomeric (sequences with 4 repetitions of 2, 3, 4 or 5 of deoxycytidines, of which telomeric or centromeric sequences natural), enter in competition with the sequence d[(CCCACA)3CCC], in particular under conditions (pH, temperature, ionic force,..), where the i-motif is observed in vitro. To identify proteins implied in association with a telomeric DNA, we used streptavidin grafted or not with the DNA sequence. We identified proteins related to DNA by mass spectrometry Maldi-tof and Q-tof MS/MS. It seems to be three proteins Yhr216p, Ylr432p and Yml056p, to which the sequences are very close. This identification was confirmed by sequencing by Edman degradation of internal peptides. The sequences of these proteins are similar (~ 78 %) with the two inosine 5'-monophosphate dehydrogenases human (IMPDH 1 and 2 (mass of ~ 56 kDa)) known for their implication in the metabolism of purins. Those are usually observed in tetrameric form (mass > 200 kDa). In spite of the presence in human nuclear extracts HeLa of one protein having native characteristics of migration identical to those observed at yeast, and different from already identified proteins ASF/SF2 and hnRNP K, we showed that proteins IMPDH 1 and 2 purified are not able to only fix human telomeric DNA. We also show that the identified proteins (Yhr216p, Ylr432p and Yml056p) bound with the complementary strand rich in G during electrophoretic migrations in the presence of candidates and show that this association is specific. To our knowledge, it seems to be the first telomeric yeast proteins interacting separately with each of the two bits under incubating conditions where we observed the formation of quadruplexes of guanines and I-motif.
Complete list of metadatas

Cited literature [179 references]  Display  Hide  Download

https://pastel.archives-ouvertes.fr/tel-00003611
Contributor : Jean-François Cornuel <>
Submitted on : Tuesday, October 21, 2003 - 2:39:16 PM
Last modification on : Saturday, November 9, 2019 - 2:08:51 AM
Long-term archiving on: : Wednesday, November 23, 2016 - 3:43:50 PM

Identifiers

  • HAL Id : tel-00003611, version 1

Citation

Jean-François Cornuel. Complexation d'un fragment de brin télomérique riche en C par des protéines de Saccharomyces cerevisiae. Identification génomique aux IMPDH. Possible repliement en motif i de l'ADN complexé.. Biochimie [q-bio.BM]. Ecole Polytechnique X, 2000. Français. ⟨tel-00003611⟩

Share

Metrics

Record views

509

Files downloads

475