Cdc48 and a ubiquitin ligase drive disassembly of the CMG helicase at the end of DNA replication

Introduction Chromosome replication is initiated by a universal mechanism in eukaryotic cells. This mechanism entails the assembly and activation at replication origins of the DNA helicase known as CMG (Cdc45-MCM-GINS), which is essential for the progression of replication forks. The replisome is built around the CMG helicase, which associates stably with DNA replication forks until the termination of DNA synthesis. The mechanism by which CMG is disassembled was unknown until now but is likely to represent a key regulated step at the end of chromosome replication. Embedded Image Regulated disassembly of the CMG helicase at the end of chromosome replication in budding yeast. It is very important that the CMG helicase is not displaced from replication forks during elongation, because it cannot be reloaded. When replication terminates, however, the ubiquitin ligase SCFDia2 and the Cdc48 segregase induce disassembly of the CMG helicase, leading to dissolution of the replisome. Rationale The CMG helicase exists only at DNA replication forks but can be isolated from extracts of S-phase budding yeast cells, after digestion of chromosomal DNA. We screened for posttranslational modifications of the CMG helicase that might regulate its function. Results Here we show that the CMG helicase is ubiquitylated during the final stages of chromosome replication in Saccharomyces cerevisiae, specifically on its Mcm7 subunit. The F-box protein Dia2 is essential in vivo for ubiquitylation of CMG, and the SCFDia2 ubiquitin ligase is also required to ubiquitylate CMG in vitro on its Mcm7 subunit in extracts of S-phase yeast cells. Ubiquitylated CMG exists only transiently in vivo, as it is rapidly disassembled in a reaction that is independent of the proteasome but requires the Cdc48/p97 segregase, which associates with ubiquitylated CMG. Consistent with these data, we show that Dia2 is essential for disassembly of the CMG helicase at the end of S phase in budding yeast. Rather than causing dissolution of the active helicase, Dia2 specifically induces the disassembly of terminated CMG complexes, which suggests that the helicase undergoes a change at the end of DNA replication, predisposing it for disassembly