Topología del ADN Nucleosomal en centrómeros y promotores genéticos de Saccharomyces Cerevisiae

Abstract

This thesis has focused on the accurate measure, in Saccharomyces cerevisiae cells, of the DNA linking number stabilized by canonical nucleosomes in vivo, as well as the centromere and several promoter regions topological contributions. To that end, TRP1ARS1 sequences, as well as its derivatives, were introduced in different S. cerevisiae strains: JCW25 (TOP1 TOP2 TOP3), JCW26 (TOP1 top2ts TOP3) y JCW27 (Δtop1 TOP2 TOP3), extracted as chromatin and analyzed by electrophoretic techniques in order to determine its in vivo topology. Thanks to the little size of these DNA ring (never bigger than 2kb), the distributions of the topoisomers have allowed to identified any modification of the chromatin structure_x000D_ Topology of TRP1ARS1:_x000D_ The TRP1ARS1 (TA1)1453 bp sequence has been cycled and used to electroporate different S. cerevisiae strains. The topological analysis of the nucleosomes located in this region has resulted in a Linking number difference (ΔLk) of -9.6 units. This value points to the stabilization of -1.37 per nucleosome, contrasting with the assumed value of -1 unit per nucleosome, known as the “linking number paradox”_x000D_ Centromere:_x000D_ S. cerevisiae centromere is characterized by 11-120 bp sequence, with three differentiated elements (CDEI, CDEII y CDEIII), as well as a hemisome (H2A+H2B+cenH3+H4) harboring the variant H3 histone CenH3. Over CDEI and CDEII sequences two protein complexes are bound, Cbf1 and CBF3 respectively, which are able to bend the DNA in vitro._x000D_ Along this thesis, the topology of centromere of chromosome IV (CEN4) of S. cerevisiae, cloned in TA1 ring, has been studied resulting in a gain of +0.6 units of ΔLk. This value has been established when comparing the topology of TA1+CEN4 with the observed in DNA rings where CDEIII of CEN4 has been mutated, preventing CBF3 bound and as a consequence, abolishing centromere function, or where CEN4 has been substituted by High2 sequence which establishes a well-positioned nucleosome._x000D_ The topological analysis of CEN4 rings of different size, in different strains of S. cerevisiae with altered topoisomerase activity, have confirmed that the stabilization of ΔLk =+0.6 is a strong and intrinsic characteristic of the centromeric complex. Besides this, the comparative study of CEN2, CEN4, CEN7 and CEN12 centromeres has discarded the stabilization of +0.6 units depending on the rotational phase of CDEI and CDEIII so, it is not due to an interaction between both protein complexes._x000D_ From these data, it can be concluded that the stabilization of ΔLk = +0.6 would be determined by a right-handed loop around the hemisome formed over CDEII or, by the mixed of different DNA curvature planes in the regions of CDEI, CDEII y CDEIII._x000D_ Promoters:_x000D_ Taking the advantage of the methodology used in the topological analysisi of the centromere, a study of promoters structure which are located in genes affected by topoisomerase II activity has just been started. It has been observed a difference between the linking number established by these promoters and a non-promoter fragment of chromatin used as a control. Furthermore, the inactivation of Topo II causes in these promoters topological changes that differ from the chromatin used as a control which suggests a trasncription regulation mechanism by Topo II.

Date of Award	23 Oct 2014
Original language	Spanish
Supervisor	Joaquim Roca Bosch (Director) & Inmaculada Ponte Marull (Tutor)