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Reduced dosage of a single fission yeast MCM protein causes genetic instability and S phase delay
D T Liang1, J A Hodson, S L Forsburg
1Department of Biology, University of California, San Diego CA 92093, USA. forsburg@salk.edu
Abstract:
MCM proteins are a conserved family of eukaryotic replication factors implicated in the initiation of DNA replication and in the discrimination between replicated and unreplicated chromatin. However, most mcm mutants in yeast arrest the cell cycle after bulk DNA synthesis has occurred. We investigated the basis for this late S phase arrest by analyzing the effects of a temperature-sensitive mutation in fission yeast cdc19(+ )(mcm2(+)). cdc19-P1 cells show a dramatic loss of viability at the restrictive temperature, which is not typical of all S phase mutants. The cdc19-P1 cell cycle arrest requires an intact damage-response checkpoint and is accompanied by increased rates of chromosome loss and mitotic recombination. Chromosomes from cdc19-P1 cells migrate aberrantly in pulsed-field gels, typical of strains arrested with unresolved replication intermediates. The cdc19-P1 mutation reduces the level of the Cdc19 protein at all temperatures. We compared the effects of disruptions of cdc19(+ )(mcm2(+)), cdc21(+ )(mcm4(+)), nda4(+ )(mcm5(+)) and mis5(+ )(mcm6(+)); in all cases, the null mutants underwent delayed S phase but were unable to proceed through the cell cycle. Examination of protein levels suggests that this delayed S phase reflects limiting, but not absent, MCM proteins. Thus, reduced dosage of MCM proteins allows replication initiation, but is insufficient for completion of S phase and cell cycle progression.
Insights
Reduced levels of minichromosome maintenance (MCM) proteins in fission yeast cause late S phase arrest. This arrest is linked to DNA damage and replication stress, preventing cell cycle progression.
Area of Science:
- Cell Biology
- Molecular Biology
- Genetics
Background:
- Minichromosome maintenance (MCM) proteins are essential eukaryotic replication factors.
- MCM proteins regulate DNA replication initiation and chromatin replication status.
- Most yeast mcm mutants arrest late in S phase after bulk DNA synthesis.
Purpose of the Study:
- Investigate the molecular basis for late S phase arrest in fission yeast.
- Analyze the effects of a temperature-sensitive mutation in cdc19+ (mcm2+).
- Determine the role of MCM protein dosage in cell cycle progression.
Main Methods:
- Utilized a temperature-sensitive mutation (cdc19-P1) in fission yeast.
- Assessed cell viability, DNA damage response, chromosome loss, and mitotic recombination.
- Analyzed chromosome migration using pulsed-field gel electrophoresis.
- Compared effects of null mutations in multiple MCM genes (cdc19+, cdc21+, nda4+, mis5+).
Main Results:
- cdc19-P1 cells exhibited loss of viability and required an intact damage-response checkpoint for arrest.
- Arrested cells showed increased chromosome loss and mitotic recombination, with aberrant chromosome migration.
- Reduced Cdc19 protein levels were observed.
- Null mutants of MCM genes displayed delayed S phase but failed to progress through the cell cycle.
- Low MCM protein levels allowed initiation but not completion of DNA replication.
Conclusions:
- Reduced MCM protein dosage is insufficient for complete S phase progression.
- Replication intermediates persist when MCM protein levels are limiting.
- MCM protein levels are critical for timely and complete DNA replication and cell cycle progression.