title: Fork rotation and DNA precatenation are restricted during DNA replication to prevent chromosomal instability.
abstract: Faithful genome duplication and inheritance require the complete resolution of all intertwines within the parental DNA duplex. This is achieved by topoisomerase action ahead of the replication fork or by fork rotation and subsequent resolution of the DNA precatenation formed. Although fork rotation predominates at replication termination, in vitro studies have suggested that it also occurs frequently during elongation. However, the factors that influence fork rotation and how rotation and precatenation may influence other replication-associated processes are unknown. Here we analyze the causes and consequences of fork rotation in budding yeast. We find that fork rotation and precatenation preferentially occur in contexts that inhibit topoisomerase action ahead of the fork, including stable protein-DNA fragile sites and termination. However, generally, fork rotation and precatenation are actively inhibited by Timeless/Tof1 and Tipin/Csm3. In the absence of Tof1/Timeless, excessive fork rotation and precatenation cause extensive DNA damage following DNA replication. With Tof1, damage related to precatenation is focused on the fragile protein-DNA sites where fork rotation is induced. We conclude that although fork rotation and precatenation facilitate unwinding in hard-to-replicate contexts, they intrinsically disrupt normal chromosome duplication and are therefore restricted by Timeless/Tipin.
authors.affiliation: Genome Damage and Stability Centre, University of Sussex, Falmer, Brighton, East Sussex BN1 9RQ, United Kingdom;
authors.forename: Stephanie A
authors.initials: SA
authors.lastname: Schalbetter
authors.affiliation: Genome Damage and Stability Centre, University of Sussex, Falmer, Brighton, East Sussex BN1 9RQ, United Kingdom;
authors.forename: Sahar
authors.initials: S
authors.lastname: Mansoubi
authors.affiliation: Genome Damage and Stability Centre, University of Sussex, Falmer, Brighton, East Sussex BN1 9RQ, United Kingdom; School of Biochemistry, University of Bristol, Bristol BS8 1TD, United Kingdom.
authors.forename: Anna L
authors.initials: AL
authors.lastname: Chambers
authors.affiliation: Genome Damage and Stability Centre, University of Sussex, Falmer, Brighton, East Sussex BN1 9RQ, United Kingdom;
authors.forename: Jessica A
authors.initials: JA
authors.lastname: Downs
authors.affiliation: Genome Damage and Stability Centre, University of Sussex, Falmer, Brighton, East Sussex BN1 9RQ, United Kingdom; jon.baxter@sussex.ac.uk.
authors.forename: Jonathan
authors.identifier: http://orcid.org/0000-0002-4455-9717
authors.initials: J
authors.lastname: Baxter
mesh_terms: D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000502:physiology*; D043171:Chromosomal Instability; D004247:DNA / Q000737:chemistry*; D004261:DNA Replication; D004250:DNA Topoisomerases, Type II / Q000378:metabolism; D004271:DNA, Fungal / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000502:physiology*; D017353:Gene Deletion; D005838:Genotype; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000502:physiology*; D004718:Saccharomycetales / Q000235:genetics; D013269:Stochastic Processes
chemical_list: D018797:Cell Cycle Proteins; C508610:Csm3p protein, S cerevisiae; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D029701:Saccharomyces cerevisiae Proteins; C119636:TOF1 protein, S cerevisiae; D004247:DNA; D004250:DNA Topoisomerases, Type II
keywords: DNA catenation; DNA replication; DNA topology; fork rotation; topoisomerases