CMG helicase disassembly is essential and mediated by two pathways in Saccharomyces cerevisiae

Abstract

The final stage of DNA replication termination involves the disassembly of the replisome, which is initiated by the ubiquitylation of the Mcm7 subunit of the CMG helicase complex. This process is driven by the ubiquitin ligases SCFDia2 in budding yeast and CUL2LRR1 in metazoa. Although the LRR1 substrate receptor is essential for viability in animal cells, it remains unclear whether this reflects the importance of CMG helicase disassembly, or else is due to a failure to ubiquitylate other CUL2LRR1 targets. Similarly, the absence of Dia2 in budding yeast cells results in profound growth defects and genome instability, but it was unclear whether these cellular phenotypes are caused by the inability to ubiquitylate the CMG helicase during DNA replication termination. This study aimed to reveal the importance of this process in Saccharomyces cerevisiae.

To explore the physiological significance of CMG ubiquitylation, I have identified and mutated ubiquitylation sites in budding yeast Mcm7 using a reconstituted ubiquitylation assay with a set of purified yeast proteins. This approach led to the generation of the mcm7-10R allele, which causes a significant defect in CMG ubiquitylation and delays CMG disassembly in budding yeast cells. Importantly, the phenotypes of mcm7-10R mirror the effects of a hypo-morphic allele of dia2, which similarly impairs CMG ubiquitylation and delays CMG helicase disassembly. Moreover, the combination of the two mutants produces a near-complete block to CMG disassembly, effectively emulating the deletion of the DIA2 gene. These findings indicate that the previously reported phenotypes of dia2∆ cells are predominantly due to the underlying defect in CMG helicase ubiquitylation and disassembly.

Crucially, this study reveals that the viability of both mcm7-10R and dia2 mutants is dependent on the two members of the Pif1 DNA helicase family in budding yeast, namely Rrm3 and Pif1. These helicases assist replication forks to overcome a range of obstacles that are encountered across the genome. My data indicate that Rrm3, and likely Pif1, preserves the viability of mcm7-10R cells by facilitating the removal of ‘old’ CMG complexes that persist from one cell cycle to the next in the absence of CMG ubiquitylation. These findings suggest that the Pif1-family of helicases provides a back-up pathway for CMG helicase disassembly in budding yeast cells, and further indicate that the removal of persistent CMG helicase complexes from chromatin is essential for cell viability.

Date of Award	2023
Original language	English
Awarding Institution	University of Dundee
Supervisor	Karim Labib (Supervisor)