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Updated: Mar 17, 2026
Genetic Screen for Identification of Multicopy Suppressors in Schizosaccharomyces pombe
Published on: September 13, 2022
Characterization of a Novel MMS-Sensitive Allele of Schizosaccharomyces pombe mcm4
Nimna S Ranatunga1, Susan L Forsburg2
1Program in Molecular and Computational Biology, University of Southern California, Los Angeles, California 90089.
Abstract:
The minichromosome maintenance (MCM) complex is the conserved helicase motor of the eukaryotic replication fork. Mutations in the Mcm4 subunit are associated with replication stress and double strand breaks in multiple systems. In this work, we characterize a new temperature-sensitive allele of Schizosaccharomyces pombe mcm4+ Uniquely among known mcm4 alleles, this mutation causes sensitivity to the alkylation damaging agent methyl methanesulfonate (MMS). Even in the absence of treatment or temperature shift, mcm4-c106 cells show increased repair foci of RPA and Rad52, and require the damage checkpoint for viability, indicating genome stress. The mcm4-c106 mutant is synthetically lethal with mutations disrupting fork protection complex (FPC) proteins Swi1 and Swi3. Surprisingly, we found that the deletion of rif1+ suppressed the MMS-sensitive phenotype without affecting temperature sensitivity. Together, these data suggest that mcm4-c106 destabilizes replisome structure.
Insights
A new mutation in the Mcm4 protein (minichromosome maintenance complex subunit 4) causes genome instability in fission yeast. This Mcm4 defect destabilizes the replication fork, leading to DNA damage and checkpoint activation.
Area of Science:
- * Molecular Biology
- * Genetics
- * Cell Biology
Background:
- * The minichromosome maintenance (MCM) complex functions as the replicative helicase in eukaryotes.
- * Mutations in the MCM4 subunit are linked to replication stress and DNA double-strand breaks.
- * Understanding MCM complex stability is crucial for comprehending genome integrity.
Purpose of the Study:
- * To characterize a novel temperature-sensitive allele of Schizosaccharomyces pombe mcm4+, designated mcm4-c106.
- * To investigate the effects of this mutation on DNA replication, genome stability, and DNA damage response pathways.
- * To explore genetic interactions and identify suppressors of the mcm4-c106 phenotype.
Main Methods:
- * Characterization of the mcm4-c106 mutant in Schizosaccharomyces pombe.
- * Assessment of sensitivity to methyl methanesulfonate (MMS), a DNA alkylation agent.
- * Analysis of DNA repair foci (RPA, Rad52) and DNA damage checkpoint activation.
- * Synthetic lethality studies with mutations in fork protection complex (FPC) proteins (Swi1, Swi3).
- * Genetic screening for suppressors of the MMS-sensitive phenotype, including deletion of rif1+.
Main Results:
- * The mcm4-c106 mutant exhibits sensitivity to MMS and spontaneous genome stress, indicated by increased RPA and Rad52 repair foci.
- * Cells with the mcm4-c106 mutation require the DNA damage checkpoint for viability.
- * Synthetic lethality was observed between mcm4-c106 and mutations in Swi1 or Swi3, highlighting the importance of fork protection.
- * Deletion of rif1+ suppressed the MMS sensitivity of mcm4-c106 without affecting its temperature sensitivity.
Conclusions:
- * The mcm4-c106 mutation destabilizes the replisome structure, leading to replication stress and genome instability.
- * This study reveals a novel role for Rif1 in suppressing MCM-related replication defects.
- * The findings provide insights into the intricate mechanisms governing replication fork stability and DNA repair.
