title: Reversible Top1 cleavage complexes are stabilized strand-specifically at the ribosomal replication fork barrier and contribute to ribosomal DNA stability.
abstract: Various topological constraints at the ribosomal DNA (rDNA) locus impose an extra challenge for transcription and DNA replication, generating constant torsional DNA stress. The topoisomerase Top1 is known to release such torsion by single-strand nicking and re-ligation in a process involving transient covalent Top1 cleavage complexes (Top1cc) with the nicked DNA. Here we show that Top1ccs, despite their usually transient nature, are specifically targeted to and stabilized at the ribosomal replication fork barrier (rRFB) of budding yeast, establishing a link with previously reported Top1 controlled nicks. Using ectopically engineered rRFBs, we establish that the rRFB sequence itself is sufficient for induction of DNA strand-specific and replication-independent Top1ccs. These Top1ccs accumulate only in the presence of Fob1 and Tof2, they are reversible as they are not subject to repair by Tdp1- or Mus81-dependent processes, and their presence correlates with Top1 provided rDNA stability. Notably, the targeted formation of these Top1ccs accounts for the previously reported broken replication forks at the rRFB. These findings implicate a novel and physiologically regulated mode of Top1 action, suggesting a mechanism by which Top1 is recruited to the rRFB and stabilized in a reversible Top1cc configuration to preserve the integrity of the rDNA.
authors.affiliation: Department of Biomedicine, University of Basel, 4058 Basel, Switzerland and Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, 4058 Basel, Switzerland.
authors.forename: Claudia
authors.initials: C
authors.lastname: Krawczyk
authors.forename: Vincent
authors.initials: V
authors.lastname: Dion
authors.forename: Primo
authors.initials: P
authors.lastname: Schär
authors.forename: Olivier
authors.initials: O
authors.lastname: Fritsch
mesh_terms: D053903:DNA Breaks, Double-Stranded; D053837:DNA Cleavage; D004261:DNA Replication; D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D004275:DNA, Ribosomal / Q000096:biosynthesis* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D055550:Protein Stability; D053484:RecQ Helicases / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D004275:DNA, Ribosomal; D004268:DNA-Binding Proteins; C105579:FOB1 protein, S cerevisiae; D047908:Intracellular Signaling Peptides and Proteins; D029701:Saccharomyces cerevisiae Proteins; C119637:TOF2 protein, S cerevisiae; C090472:SGS1 protein, S cerevisiae; D053484:RecQ Helicases; D004264:DNA Topoisomerases, Type I