Molecular Mechanism of DNA Topoisomerase I-Dependent rDNA Silencing: Sir2p Recruitment at Ribosomal Genes

Saccharomyces cerevisiae sir2Δ or top1Δ mutants exhibit similar phenotypes involving ribosomal DNA, including (i) loss of transcriptional silencing, resulting in non-coding RNA hyperproduction from cryptic RNA polymerase II promoters; (ii) alterations in recombination; and (iii) a general increase in histone acetylation. Given the distinct enzymatic activities of Sir2 and Top1 proteins, a histone deacetylase and a DNA topoisomerase, respectively, we investigated whether genetic and/or physical interactions between the two proteins could explain the shared ribosomal RNA genes (rDNA) phenotypes. We employed an approach of complementing top1Δ cells with yeast, human, truncated, and chimeric yeast/human TOP1 constructs and of assessing the extent of non-coding RNA silencing and histone H4K16 deacetylation. Our findings demonstrate that residues 115–125 within the yeast Top1p N-terminal domain are required for the complementation of the top1Δ rDNA phenotypes. In chromatin immunoprecipitation and co-immunoprecipitation experiments, we further demonstrate the physical interaction between Top1p and Sir2p. Our genetic and biochemical studies support a model whereby Top1p recruits Sir2p to the rDNA and clarifies a structural role of DNA topoisomerase I in the epigenetic regulation of rDNA, independent of its known catalytic activity.