In vivo studies of the non-transcribed spacer region of rDNA in Saccharomyces cerevisiae

The rDNA is the genetic locus that encodes the ribosomal RNAs and physicall y defines where ribosomes start to assemble. In the yeast Saccharomyces cer evisiae, its highly repetitive structure makes it a very interesting target for studies about genome stability, chromatin mediated transcriptional sil encing and progression of aging. In fact, recombination among the repeated units is suppressed in a WT cell. Moreover, when genes transcribed by RNA p olymerase II are inserted in the rDNA cluster, their transcription is silen ced. Finally, the formation of rDNA minicircles has been shown to be one of the causes of aging in yeast. DNA topoisomerase I has been shown to suppre ss 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 mol ecular basis of how this enzyme interferes with these functions is yet unkn own. Here are reported results obtained by in vivo studies of DNA-protein i nteractions occurring on the rDNA locus. The analyses include mapping of: n ucleosome positioning; RNA polymerase I transcription factors and DNA topoi somerase I cleavage sites. Important conclusions can be drawn: nucleosome p ositioning in the Non-transcribed Spacer (NTS) is not affected by RNA polym erase I transcription; the RNA polymerase I transcription factors bind DNA in vivo with a defined hierarchy, the DNA topoisomerase I cleaves the NTS i n very specific sites, but cleavage is not induced by RNA polymerase I tran scription. These in vivo studies help to characterize, the molecular basis of important phenomena as the transcriptional silencing and genome stabilit y in yeast.