DNA topoisomerase I activity on the rDNA chromatin

Gruppo di Ricerca: 
Anna D'Alfonso, Francesca Di Felice, Valentina Carlini e Giorgio Camilloni

In eukaryotic nuclei DNA topology is regulated by specific enzymes: the DNA topoisomerases (Wang, 1996). These enzymes are divided into different classes depending on their catalytic activity. Among these proteins, the eukaryotic type I DNA topoisomerase controls DNA topology by transiently breaking and resealing one strand of DNA. In eukaryotes, DNA topoisomerase I is involved in DNA metabolism and functions during transcription and replication, in order to reduce the torsional stress deriving from these activities. In addition, this enzyme has been shown to be part of transcriptional machinery and in the absence of DNA topoisomerase I, rDNA silencing is abolished.
We investigated the DNA Topoisomerase 1 reactivity in cells whose chromatin apparatus is altered. This result could be obtained by reducing histone content or increasing histone acetylation levels and gives interesting evidences concerning the capability of DNA topoisomerase 1 to react on chromatin substrates. Both conditions determine an accessible chromatin structure, that in principle, could facilitate Topo 1 reaction with DNA. Our recent studies on histone content in yeast and mammal cells, gave comparable results on basic chromatin structure (Celona et al., 2011). The model system utilized is Saccharomyces cerevisiae.
We evaluated the whole chromatin organization of the rDNA area. MNase accessibility was measured by end-labelling technique after in vivo digestion of yeast nuclei with MNase (Vogelauer et al., 1998). The digestion patterns obtained clarifies the nucleosome organization of the region encompassing the 35SRNA termination site and the replication fork barrier (RFB) sequence. In order to map with higher detail the region where MNAse accessibility is evaluated, we further studied the RFB region. We verified the specific nucleosome occupancy in fob1 mutants at the RFB region. MNase-PCR experiments were performed. The results obtained comparing nucleosome occupancy in WT and fob1 strain, at the RFB sequence, indicate that in fob1 mutant nucleosomes show altered organization. This suggests that chromatin structure requires Fob1p presence.
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