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Summary: Eukaryotic cells contain multiple versions of the E3 ubiquitin ligase known as the SCF (Skp1/cullin/F box), each of which is distinguished by a different F box protein that uses a domain at the carboxyl terminus to recognize substrates . The F box protein Dia2 is an important determinant of genome stability in budding yeast , but its mode of action is poorly understood. Here we show that SCFDia2 associates with the replisome progression complex (RPC) that assembles around the MCM2-7 helicase at DNA replication forks . This interaction requires the RPC components Mrc1 and Ctf4, both of which associate with a tetratricopeptide repeat (TPR) domain located at the amino terminus of Dia2. Our data indicate that the TPR domain of Dia2 tethers SCFDia2 to the RPC, probably increasing the local concentration of the ligase at DNA replication forks. This regulation becomes important in cells that accumulate stalled DNA replication forks at protein-DNA barriers, perhaps aiding the interaction of SCFDia2 with key substrates. Our findings suggest that the amino-terminal domains of other F box proteins might also play an analogous regulatory role, controlling the localization of the cognate SCF complexes. [Copyright &y& Elsevier]

An excerpt is presented of an article printed online at http://dx.doi.org/10.1126/science.1253596 which discusses the Cdc45-MCM-GINS (CMG) helicase, its role in stabilizing DNA replication forks, and its disassembly following termination of DNA replication.

The exploitation of synthetic lethality by small-molecule targeting of pathways that maintain genomic stability is an attractive chemotherapeutic approach. The Ctf4/AND-1 protein hub, which links DNA replication, repair, and chromosome segregation, represents a novel target for the synthetic lethality approach. Herein, we report the design, optimization, and validation of double-click stapled peptides encoding the Ctf4-interacting peptide (CIP) of the replicative helicase subunit Sld5. By screening stapling positions in the Sld5 CIP, we identified an unorthodox i,i+6 stapled peptide with improved, submicromolar binding to Ctf4. The mode of interaction with Ctf4 was confirmed by a crystal structure of the stapled Sld5 peptide bound to Ctf4. The stapled Sld5 peptide was able to displace the Ctf4 partner DNA polymerase α from the replisome in yeast extracts. Our study provides proof-of-principle evidence for the development of small-molecule inhibitors of the human CTF4 orthologue AND-1., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Replisome disassembly is the final step of DNA replication in eukaryotes, involving the ubiquitylation and CDC48-dependent dissolution of the CMG helicase (CDC45-MCM-GINS). Using Caenorhabditis elegans early embryos and Xenopus laevis egg extracts, we show that the E3 ligase CUL-2 LRR-1 associates with the replisome and drives ubiquitylation and disassembly of CMG, together with the CDC-48 cofactors UFD-1 and NPL-4. Removal of CMG from chromatin in frog egg extracts requires CUL2 neddylation, and our data identify chromatin recruitment of CUL2 LRR1 as a key regulated step during DNA replication termination. Interestingly, however, CMG persists on chromatin until prophase in worms that lack CUL-2 LRR-1 , but is then removed by a mitotic pathway that requires the CDC-48 cofactor UBXN-3, orthologous to the human tumour suppressor FAF1. Partial inactivation of lrr-1 and ubxn-3 leads to synthetic lethality, suggesting future approaches by which a deeper understanding of CMG disassembly in metazoa could be exploited therapeutically.

Checkpoints were originally identified as signalling pathways that delay mitosis in response to DNA damage or defects in chromosome replication, allowing time for DNA repair to occur. The ATR (ataxia- and rad-related) and ATM (ataxia-mutated) protein kinases are recruited to defective replication forks or to sites of DNA damage, and are thought to initiate the DNA damage response in all eukaryotes. In addition to delaying cell cycle progression, however, the S-phase checkpoint pathway also controls chromosome replication and DNA repair pathways in a highly complex fashion, in order to preserve genome integrity. Much of our understanding of this regulation has come from studies of yeasts, in which the best-characterized targets are the stimulation of ribonucleotide reductase activity by multiple mechanisms, and the inhibition of new initiation events at later origins of DNA replication. In addition, however, the S-phase checkpoint also plays a more enigmatic and apparently critical role in preserving the functional integrity of defective replication forks, by mechanisms that are still understood poorly. This review considers some of the key experiments that have led to our current understanding of this highly complex pathway.