title: Tethering of SCF(Dia2) to the Replisome Promotes Efficient Ubiquitylation and Disassembly of the CMG Helicase.
abstract: Disassembly of the Cdc45-MCM-GINS (CMG) DNA helicase, which unwinds the parental DNA duplex at eukaryotic replication forks, is the key regulated step during replication termination but is poorly understood. In budding yeast, the F-box protein Dia2 drives ubiquitylation of the CMG helicase at the end of replication, leading to a disassembly pathway that requires the Cdc48 segregase. The substrate-binding domain of Dia2 comprises leucine-rich repeats, but Dia2 also has a TPR domain at its amino terminus that interacts with the Ctf4 and Mrc1 subunits of the replisome progression complex, which assembles around the CMG helicase at replication forks. Previous studies suggested two disparate roles for the TPR domain of Dia2, either mediating replisome-specific degradation of Mrc1 and Ctf4 or else tethering SCF(Dia2) (SCF [Skp1/cullin/F-box protein]) to the replisome to increase its local concentration at replication forks. Here, we show that SCF(Dia2) does not mediate replisome-specific degradation of Mrc1 and Ctf4, either during normal S phase or in response to replication stress. Instead, the tethering of SCF(Dia2) to the replisome progression complex increases the efficiency of ubiquitylation of the Mcm7 subunit of CMG, both in vitro and in vivo. Correspondingly, loss of tethering reduces the efficiency of CMG disassembly in vivo and is synthetic lethal in combination with a disassembly-defective allele of CDC48. Residual ubiquitylation of Mcm7 in dia2-ΔTPR cells is still CMG specific, highlighting the complex regulation of the final stages of chromosome replication, about which much still remains to be learned.
authors.affiliation: Cancer Research UK Manchester Institute, University of Manchester, Wilmslow Road, Manchester M20 4BX, UK.
authors.forename: Timurs
authors.initials: T
authors.lastname: Maculins
authors.affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit, Sir James Black Centre, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK.
authors.forename: Pedro Junior
authors.initials: PJ
authors.lastname: Nkosi
authors.affiliation: Cancer Research UK Manchester Institute, University of Manchester, Wilmslow Road, Manchester M20 4BX, UK.
authors.forename: Hiroko
authors.initials: H
authors.lastname: Nishikawa
authors.affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit, Sir James Black Centre, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK. Electronic address: kpmlabib@dundee.ac.uk.
authors.forename: Karim
authors.initials: K
authors.lastname: Labib
mesh_terms: D004261:DNA Replication; D004271:DNA, Fungal / Q000235:genetics* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D044783:F-Box Proteins / Q000235:genetics* / Q000378:metabolism; D064168:Minichromosome Maintenance Complex Component 7 / Q000235:genetics* / Q000378:metabolism; D016196:S Phase; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism; D054875:Ubiquitination
chemical_list: C078338:CTF4 protein, S cerevisiae; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; C495983:Dia2 protein, S cerevisiae; D044783:F-Box Proteins; D029701:Saccharomyces cerevisiae Proteins; C491088:MCM7 protein, S cerevisiae; D064168:Minichromosome Maintenance Complex Component 7