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:DNA replication initiation requires activation of the CMG helicase to establish the replisome. This process involves the extrusion of single-stranded DNA (ssDNA) from the central channel of MCM double hexamers, allowing the two CMG helicases to pass each other; however, the factors that mediate this process in human cells remain unclear. We show that degron-mediated depletion of either MCM10 or RECQL4 alone causes only mild replication defects, whereas simultaneous depletion of both proteins completely blocks CMG activation. ChIP-seq analyses demonstrate that RECQL4 localises to replication initiation zones (IZs) independently of MCM10, whereas MCM10 recruitment to IZs is enhanced upon RECQL4 depletion, suggesting RECQL4 primarily functions in CMG activation, and MCM10 acts as a backup or supporting factor. Rescue experiments further indicate that RECQL4 cooperates with MCM10 through direct interaction, and that their ssDNA-binding activity underlies their functional overlap. We propose MCM10 and RECQL4 act cooperatively and redundantly to promote CMG activation.
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.