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A conserved Polϵ binding module in Ctf18-RFC is required for S-phase checkpoint activation downstream of Mec1.García-Rodríguez, Luis J; De Piccoli, G; Marchesi, Vanessa; Jones, R; Edmondson, R; Labib, K; Cancer Research UK Manchester Institute, University of Manchester, Wilmslow Road, Manchester M20 4BX (2015-08-06)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.
Tethering of SCF(Dia2) to the replisome promotes efficient ubiquitylation and disassembly of the CMG helicase.Maculins, Timurs; Nkosi, P; Nishikawa, H; Labib, K; Cancer Research UK Manchester Institute, University of Manchester, Wilmslow Road, Manchester M20 4BX, UK (2015-08-31)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 [1, 2]. 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 [4, 5], 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.