Abstract: The rDNA cluster is the genetic locus encoding the ribosomal RNAs and physically defines where ribosomes begin to be assembled. In the yeast Saccharomyces cerevisiae, the highly repetitive structure of this locus makes it a very interesting target for studies about genome stability, chromatin-mediated transcriptional silencing and progression of aging. In fact, recombination among the repeated units is suppressed in a WT cell. Moreover, when genes transcribed by RNA polymerase II are inserted in the rDNA cluster, their transcription is silenced. Finally, the formation of rDNA minicircles (ERCs) has been shown to be one of the causes of aging in yeast. DNA topoisomerase I have been shown to suppress recombination specifically at the rDNA of S.cerevisiae. Moreover, also the chromatin structure of this locus is affected in a top1 strain, because rDNA specific transcriptional silencing is abolished. Nonetheless, the molecular basis of how this enzyme interferes with these functions is yet unknown. Here are reported results obtained by in vivo studies of DNA protein interactions occurring on the rDNA locus. The analyses include a fine mapping of nucleosome positioning; RNA polymerase I transcription factors and DNA topoisomerase I cleavage sites. Important conclusions can be drawn: i) nucleosome positioning in the Non Transcribed Spacer is not affected by RNA polymerase I transcription; ii) the RNA polymerase I transcription factors bind DNA in vivo with a defined hierarchy; iii) the DNA topoisomerase I cleaves the NTS in very specific sites, but cleavage is not induced by RNA polymerase I transcription. These in vivo studies help to characterize the molecular basis of important phenomena as the transcriptional silencing and genome stability in yeast.