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A mutant that affects the function of autonomously replicating sequences in yeast
Abstract:
We previously reported the isolation of a series of mcm mutants that are defective in the maintenance of minichromosomes in yeast. These minichromosomes are circular plasmids, each containing an autonomously replicating sequence (ARS) and a centromere. One of the mcm mutants, mcm2, has the following phenotype: at room temperature it affects the stability of only some minichromosomes depending on the ARS present, while at high temperature it affects all minichromosomes tested irrespective of the ARS present. Here we show that the mcm defect as well as its temperature-dependent specificity for ARSs can be demonstrated with circular as well as linear plasmids that do not contain centromeric sequences. Larger chromosomes containing multiple ARSs are also unstable in this mutant. Further analyses indicate that the mcm2 mutation causes the loss, rather than the aberrant segregation, of the circular minichromosomes. In addition, this mutation appears to stimulate mitotic recombination frequencies. These properties of the mcm2 mutant are consistent with the idea that the mcm2 mutation results in a defect in the initiation of DNA replication at ARSs, the putative chromosomal replication origins in yeast.
Insights
The mcm2 mutation in yeast disrupts minichromosome stability by affecting DNA replication initiation at autonomously replicating sequences (ARS). This defect is temperature-dependent and leads to plasmid loss and increased recombination.
Area of Science:
- Molecular Biology
- Yeast Genetics
- DNA Replication
Background:
- Minichromosomes are essential for studying DNA replication and stability in yeast.
- Minichromosome maintenance (mcm) mutants were previously identified as defective in plasmid stability.
Purpose of the Study:
- To investigate the role of the mcm2 mutation in yeast minichromosome stability.
- To determine the effect of the mcm2 mutation on autonomously replicating sequences (ARS) and chromosomal elements.
Main Methods:
- Phenotypic analysis of mcm2 mutant yeast strains carrying various minichromosomes and plasmids.
- Assessment of plasmid stability, segregation, and mitotic recombination frequencies.
- Temperature-shift experiments to evaluate the temperature-dependent nature of the mcm2 defect.
Main Results:
- The mcm2 mutation destabilizes minichromosomes, affecting both circular and linear plasmids lacking centromeres.
- The defect is temperature-dependent and impacts all tested autonomously replicating sequences (ARS) at high temperatures.
- mcm2 mutation leads to minichromosome loss and increased mitotic recombination, suggesting a defect in replication initiation.
Conclusions:
- The mcm2 mutation impairs the initiation of DNA replication at ARS elements, which function as chromosomal replication origins.
- This replication initiation defect underlies the observed minichromosome instability and increased recombination in mcm2 mutants.