title: A conserved Polϵ binding module in Ctf18-RFC is required for S-phase checkpoint activation downstream of Mec1.
abstract: Defects during chromosome replication in eukaryotes activate a signaling pathway called the S-phase checkpoint, which produces a multifaceted response that preserves genome integrity at stalled DNA replication forks. Work with budding yeast showed that the 'alternative clamp loader' known as Ctf18-RFC acts by an unknown mechanism to activate the checkpoint kinase Rad53, which then mediates much of the checkpoint response. Here we show that budding yeast Ctf18-RFC associates with DNA polymerase epsilon, via an evolutionarily conserved 'Pol ϵ binding module' in Ctf18-RFC that is produced by interaction of the carboxyl terminus of Ctf18 with the Ctf8 and Dcc1 subunits. Mutations at the end of Ctf18 disrupt the integrity of the Pol ϵ binding module and block the S-phase checkpoint pathway, downstream of the Mec1 kinase that is the budding yeast orthologue of mammalian ATR. Similar defects in checkpoint activation are produced by mutations that displace Pol ϵ from the replisome. These findings indicate that the association of Ctf18-RFC with Pol ϵ at defective replication forks is a key step in activation of the S-phase checkpoint.
authors.affiliation: Cancer Research UK Manchester Institute, University of Manchester, Wilmslow Road, Manchester M20 4BX, UK.
authors.forename: Luis J
authors.initials: LJ
authors.lastname: García-Rodríguez
authors.affiliation: Division of Biomedical Cell Biology, Warwick Medical School, University of Warwick, Coventry CV4 7AL, UK.
authors.forename: Giacomo
authors.initials: G
authors.lastname: De Piccoli
authors.affiliation: Cancer Research UK Manchester Institute, University of Manchester, Wilmslow Road, Manchester M20 4BX, UK.
authors.forename: Vanessa
authors.initials: V
authors.lastname: Marchesi
authors.affiliation: MS Bioworks, 3950 Varsity Drive, Ann Arbor, MI 48108, USA.
authors.forename: Richard C
authors.initials: RC
authors.lastname: Jones
authors.affiliation: Myeloma Institute for Research and Therapy, University of Arkansas for Medical Sciences, 4301 W Markham #776, Little Rock, AR 72205, USA.
authors.forename: Ricky D
authors.initials: RD
authors.lastname: Edmondson
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 kpmlabib@dundee.ac.uk.
authors.forename: Karim
authors.initials: K
authors.lastname: Labib
mesh_terms: D004257:DNA Polymerase II / Q000378:metabolism*; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D009097:Multienzyme Complexes / Q000378:metabolism; D009154:Mutation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D051818:Replication Protein C / Q000378:metabolism*; D059807:S Phase Cell Cycle Checkpoints; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D000099272:Replisomes
chemical_list: D004257:DNA Polymerase II; D004268:DNA-Binding Proteins; D004259:DNA-Directed DNA Polymerase; D047908:Intracellular Signaling Peptides and Proteins; D009097:Multienzyme Complexes; D017346:Protein Serine-Threonine Kinases; D051818:Replication Protein C; D029701:Saccharomyces cerevisiae Proteins; D000099272:Replisomes; C092693:CTF18 protein, S cerevisiae; C429769:Dcc1 protein, S cerevisiae; C088937:MEC1 protein, S cerevisiae