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

• top1 and sir2 share silencing loss and H4K16 hyperacetylation at rDNA.
• Top1p recruits Sir2p to ribosomal genes by interacting with its N-terminal domain.
• Top1p–Sir2p interaction justifies shared rDNA phenotypes.
• The Sir2p–Top1p interaction does not require Topo1 catalytic activity.
• New role for Topo1 as scaffold protein for molecular interactions

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 top1 ∆ 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.

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