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Título: | Genome-wide Analyses of DNA Topology and Topoisomerase Function |
Autor: | Bermúdez, Ignacio CSIC; Roca, Joaquim CSIC ORCID | Fecha de publicación: | 23-jul-2008 | Citación: | Topo2008: DNA Topoisomerases in Biology and Medicine (2008) | Resumen: | Psoralens are planar triciclic compounds than can intercalate into duplex DNA. Intercalation occurs mostly in protein-free regions and is highly sensitive to the helical tension of DNA. Upon UV irradiation, psoralen-DNA interactions become covalent and crosslink complementary DNA strands. Our objective is to obtain full chromosomal profiles of DNA topology by mapping the distribution of psoralen-mediated DNA crosslinks along the genome of s. cerevisiae; and next, analyse how these profiles change in topoisomerase mutants and how they correlate with other functional or structural elements of the genome. We incubated yeast cells with psoralen and irradiated them to produce few crosslinks (1/10 kb) into chromosomal DNA. Crosslinked fragments were then selected by sequential exonuclease digestions and radiolabelled for hybridization in genomic DNA arrays. The results showed that the genomic distribution of crosslinks produced in vivo notably differed from that obtained in vitro with protein-free topologically unconstrained genomic DNA. In addition, numerous chromosomal regions had crosslinking patterns significantly deviated from those in the global distribution. These regions likely reflect distinct topological or structural chromatin domains. Inactivation of topoisomerase II in dtop1 top2-ts cells produced drastic changes of DNA crosslinking in particular domains of chromosomes IV and IX, which appeared to be confined by specific boundary elements. When psoralen-DNA crosslinking profiles were compared with genomic run-on profiles (GRO), carried out in the same cells, no systematic correlations were found with transcriptional activity. These studies revealed, however, that all genes down regulated by topoisomerase II inactivation share a singular promoter architecture. They have a well-positioned nucleosome upstream the transcription start site. In conclusion, genome-wide analyses of DNA topology and of topoisomerase function developed in our laboratory provide novel insights to unravel mechanisms for high order chromatin organization and regulation of gene expression. | URI: | http://hdl.handle.net/10261/118068 |
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