Project description:Disassembly of the DNA helicase known as CMG (Cdc45-Mcm-GINS) is the key regulated step during DNA replication termination in eukaryotes. In budding yeast, CMG disassembly is initiated by the ubiquitin ligase SCF-Dia2, which ubiquitylates the Mcm7 subunit of CMG, leading to recruitment of the Cdc48 unfoldase. Here we have investigated the partners of the F-box protein Dia2 and tested which ones are dependent upon the TPR domain of Dia2, using yeast strains expressing ProteinA-tagged Dia2 or Dia2∆TPR. Controls include a strain expressing the TAP tag alone, and a strain expressing TAP-tagged Sld5 subunit of the CMG helicase.
Project description:Assembly of the DNA helicase known as CMG (CDC45-MCM-GINS) is the key regulated step during DNA replication initiation in eukaryotes. Using the Caenorhabditis elegans embryo as a model system, we identify a new CMG assembly factor called DNSN-1, which associates with the BRCT-domain protein MUS-101. We show that DNSN-1 is required to recruit the GINS complex to chromatin and find that DNSN-1 positions GINS on the MCM-2-7 helicase motor, by direct binding of DNSN-1 to GINS and MCM-3, on interfaces that are important for initiation and essential for viability.