title: A Novel Rrm3 Function in Restricting DNA Replication via an Orc5-Binding Domain Is Genetically Separable from Rrm3 Function as an ATPase/Helicase in Facilitating Fork Progression.
abstract: In response to replication stress cells activate the intra-S checkpoint, induce DNA repair pathways, increase nucleotide levels, and inhibit origin firing. Here, we report that Rrm3 associates with a subset of replication origins and controls DNA synthesis during replication stress. The N-terminal domain required for control of DNA synthesis maps to residues 186-212 that are also critical for binding Orc5 of the origin recognition complex. Deletion of this domain is lethal to cells lacking the replication checkpoint mediator Mrc1 and leads to mutations upon exposure to the replication stressor hydroxyurea. This novel Rrm3 function is independent of its established role as an ATPase/helicase in facilitating replication fork progression through polymerase blocking obstacles. Using quantitative mass spectrometry and genetic analyses, we find that the homologous recombination factor Rdh54 and Rad5-dependent error-free DNA damage bypass act as independent mechanisms on DNA lesions that arise when Rrm3 catalytic activity is disrupted whereas these mechanisms are dispensable for DNA damage tolerance when the replication function is disrupted, indicating that the DNA lesions generated by the loss of each Rrm3 function are distinct. Although both lesion types activate the DNA-damage checkpoint, we find that the resultant increase in nucleotide levels is not sufficient for continued DNA synthesis under replication stress. Together, our findings suggest a role of Rrm3, via its Orc5-binding domain, in restricting DNA synthesis that is genetically and physically separable from its established catalytic role in facilitating fork progression through replication blocks.
authors.affiliation: Department of Cell Biology, Microbiology, and Molecular Biology, University of South Florida, Tampa, Florida, United States of America.|Graduate Program in Cellular and Molecular Biology, University of South Florida, Tampa, Florida, United States of America.
authors.forename: Salahuddin
authors.initials: S
authors.lastname: Syed
authors.affiliation: Center for Healthy Aging, Department of Cellular and Molecular Medicine, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Claus
authors.initials: C
authors.lastname: Desler
authors.affiliation: Center for Healthy Aging, Department of Cellular and Molecular Medicine, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Lene J
authors.initials: LJ
authors.lastname: Rasmussen
authors.affiliation: Department of Cell Biology, Microbiology, and Molecular Biology, University of South Florida, Tampa, Florida, United States of America.|Cancer Biology and Evolution Program, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida, United States of America.
authors.forename: Kristina H
authors.initials: KH
authors.lastname: Schmidt
mesh_terms: D000251:Adenosine Triphosphatases / Q000235:genetics; D002453:Cell Cycle / Q000235:genetics; D018797:Cell Cycle Proteins / Q000235:genetics; D002455:Cell Division / Q000235:genetics; D002843:Chromatin / Q000235:genetics; D004247:DNA / Q000096:biosynthesis* / Q000235:genetics; D004249:DNA Damage / Q000235:genetics; D004265:DNA Helicases / Q000235:genetics*; D004261:DNA Replication / Q000235:genetics*; D026942:DNA Topoisomerases / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics; D051738:Origin Recognition Complex / Q000235:genetics*; D017354:Point Mutation; D018741:Replication Origin / Q000235:genetics; D059807:S Phase Cell Cycle Checkpoints / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics*
chemical_list: D018797:Cell Cycle Proteins; D002843:Chromatin; D004268:DNA-Binding Proteins; C441579:MRC1 protein, S cerevisiae; C497220:ORC5 protein, S cerevisiae; D051738:Origin Recognition Complex; D029701:Saccharomyces cerevisiae Proteins; D004247:DNA; D000251:Adenosine Triphosphatases; C076225:RAD5 protein, S cerevisiae; C459081:Rrm3 protein, S cerevisiae; D004265:DNA Helicases; D026942:DNA Topoisomerases; C541336:RDH54 protein, S cerevisiae