title: Rad53-Mediated Regulation of Rrm3 and Pif1 DNA Helicases Contributes to Prevention of Aberrant Fork Transitions under Replication Stress.
abstract: Replication stress activates the Mec1(ATR) and Rad53 kinases. Rad53 phosphorylates nuclear pores to counteract gene gating, thus preventing aberrant transitions at forks approaching transcribed genes. Here, we show that Rrm3 and Pif1, DNA helicases assisting fork progression across pausing sites, are detrimental in rad53 mutants experiencing replication stress. Rrm3 and Pif1 ablations rescue cell lethality, chromosome fragmentation, replisome-fork dissociation, fork reversal, and processing in rad53 cells. Through phosphorylation, Rad53 regulates Rrm3 and Pif1; phospho-mimicking rrm3 mutants ameliorate rad53 phenotypes following replication stress without affecting replication across pausing elements under normal conditions. Hence, the Mec1-Rad53 axis protects fork stability by regulating nuclear pores and DNA helicases. We propose that following replication stress, forks stall in an asymmetric conformation by inhibiting Rrm3 and Pif1, thus impeding lagging strand extension and preventing fork reversal; conversely, under unperturbed conditions, the peculiar conformation of forks encountering pausing sites would depend on active Rrm3 and Pif1.
authors.affiliation: IFOM (Fondazione Istituto FIRC di Oncologia Molecolare), Via Adamello 16, 20139 Milan, Italy; Dipartimento di Bioscienze, Università degli Studi di Milano, Via Celoria 26, 20133 Milan, Italy.
authors.forename: Silvia Emma
authors.initials: SE
authors.lastname: Rossi
authors.affiliation: IFOM (Fondazione Istituto FIRC di Oncologia Molecolare), Via Adamello 16, 20139 Milan, Italy; Dipartimento di Bioscienze, Università degli Studi di Milano, Via Celoria 26, 20133 Milan, Italy.
authors.forename: Arta
authors.initials: A
authors.lastname: Ajazi
authors.affiliation: IFOM (Fondazione Istituto FIRC di Oncologia Molecolare), Via Adamello 16, 20139 Milan, Italy.
authors.forename: Walter
authors.initials: W
authors.lastname: Carotenuto
authors.affiliation: IFOM (Fondazione Istituto FIRC di Oncologia Molecolare), Via Adamello 16, 20139 Milan, Italy; Dipartimento di Bioscienze, Università degli Studi di Milano, Via Celoria 26, 20133 Milan, Italy. Electronic address: marco.foiani@ifom.eu.
authors.forename: Marco
authors.initials: M
authors.lastname: Foiani
authors.affiliation: IFOM (Fondazione Istituto FIRC di Oncologia Molecolare), Via Adamello 16, 20139 Milan, Italy; Dipartimento di Bioscienze, Università degli Studi di Milano, Via Celoria 26, 20133 Milan, Italy. Electronic address: michele.giannattasio@ifom.eu.
authors.forename: Michele
authors.initials: M
authors.lastname: Giannattasio
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D018797:Cell Cycle Proteins / Q000235:genetics* / Q000378:metabolism; D064447:Checkpoint Kinase 2 / Q000235:genetics* / Q000378:metabolism; D004265:DNA Helicases / Q000235:genetics* / Q000378:metabolism; D004261:DNA Replication; D004271:DNA, Fungal / Q000235:genetics* / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D022022:Nuclear Pore / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D018797:Cell Cycle Proteins; D004271:DNA, Fungal; D047908:Intracellular Signaling Peptides and Proteins; D029701:Saccharomyces cerevisiae Proteins; D064447:Checkpoint Kinase 2; C088937:MEC1 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases; C067022:RAD53 protein, S cerevisiae; C085463:PIF1 protein, S cerevisiae; C459081:Rrm3 protein, S cerevisiae; D004265:DNA Helicases