Abstract
BackgroundBy screening a plasmid library for proteins that could cause silencing when targeted to the HMR locus in Saccharomyces cerevisiae, we previously reported the identification of Rtt107/Esc4 based on its ability to establish silent chromatin. In this study we aimed to determine the mechanism of Rtt107/Esc4 targeted silencing and also learn more about its biological functions.ResultsTargeted silencing by Rtt107/Esc4 was dependent on the SIR genes, which encode obligatory structural and enzymatic components of yeast silent chromatin. Based on its sequence, Rtt107/Esc4 was predicted to contain six BRCT motifs. This motif, originally identified in the human breast tumor suppressor gene BRCA1, is a protein interaction domain. The targeted silencing activity of Rtt107/Esc4 resided within the C-terminal two BRCT motifs, and this region of the protein bound to Sir3 in two-hybrid tests. Deletion of RTT107/ESC4 caused sensitivity to the DNA damaging agent MMS as well as to hydroxyurea. A two-hybrid screen showed that the N-terminal BRCT motifs of Rtt107/Esc4 bound to Slx4, a protein previously shown to be involved in DNA repair and required for viability in a strain lacking the DNA helicase Sgs1. Like SLX genes, RTT107ESC4 interacted genetically with SGS1; esc4Δ sgs1Δ mutants were viable, but exhibited a slow-growth phenotype and also a synergistic DNA repair defect.ConclusionRtt107/Esc4 binds to the silencing protein Sir3 and the DNA repair protein Slx4 via different BRCT motifs, thus providing a bridge linking silent chromatin to DNA repair enzymes.
Highlights
By screening a plasmid library for proteins that could cause silencing when targeted to the HMR locus in Saccharomyces cerevisiae, we previously reported the identification of Rtt107/ Esc4 based on its ability to establish silent chromatin
This loss of silencing at hmr::URA3 leads to no growth on 5-fluororotic acid (5-FOA) media. (C) When the strain in (B) is transformed with a Gal4 DNA binding domain (GBD) plasmid (GBD-X) capable of causing targeted silencing, this leads to restoration of silencing at hmr::URA3 and growth on 5-FOA
The targeted silencing activity was primarily due to the C-terminal two tandem BRCT motifs in Esc4, which bound to Sir3, probably through a direct interaction
Summary
By screening a plasmid library for proteins that could cause silencing when targeted to the HMR locus in Saccharomyces cerevisiae, we previously reported the identification of Rtt107/ Esc based on its ability to establish silent chromatin. Transcriptional silencing in the budding yeast Saccharomyces cerevisiae occurs at the silent mating-type loci HMR and HML, telomeres, and at the rDNA locus. At all of these silenced regions, DNA binding proteins recognize specific motifs and recruit a silencing protein complex (reviewed in [1]). Rap contributes a critical DNA-binding function, binding to the TG1–3 repeats. At both the silent mating loci and at telomeres, the DNAbinding proteins recruit a Sir protein complex that can spread to silence genes at a distance (reviewed in [2]). At HMR-E, for example, this is achieved by ORC recruitment (page number not for citation purposes)
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