Site-specific in vivo cleavages by DNA topoisomerase I in the regulatory regions of the 35 S rRNA in Saccharomyces cerevisiae are transcription independent

Eukaryotic type I DNA topoisomerase controls DNA topology by transiently br eaking and resealing one strand of DNA at a time. During transcription and replication its action reduces the torsional stress derived from these acti vities. The association of DNA topoisomerase I with the nucleolus has been reported and this enzyme was shown to be involved in yeast rDNA metabolism. Here, we have investigated the in vivo presence of DNA topoisomerase I clea vage sites in the non-transcribed spacer of the rDNA cluster. We show a spe cific profile of highly localized cleavage in relevant areas of this region . The sites are detected in the promoter and in the enhancer regions of the 35 S gene. The analysis of mutants in which transcription is prevented and /or reduced, namely a strain lacking the 43 kDa subunit of RNA polymerase I , a second one that does note transcribe, lacking a subunit of the core fac tor and another member of the RNA polymerase I transcription factors lackin g one of the UAF component which transcribes at very low level, show that D NA topoisomerase I cleavage sites are not related to transcription by RNA p olymerase I. These findings point to a role for DNA topoisomerase I that is additional to the commonly recognized function in removing the transcripti on-induced topological stress. (C) 1999 Academic Press.