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The mcm17 mutation of yeast shows a size-dependent segregational defect of a mini-chromosome
Abstract:
Mini-chromosome-maintenance (mcm) mutants were described earlier as yeast mutants which could not stably maintain mini-chromosomes. Out of these, the ARS-specific class has been more extensively studied and is found to lose chromosomes and mini-chromosomes due to a defect in the initiation of DNA replication at yeast ARSs. In the present study we have identified a number of mcm mutants which show size-dependent loss of mini-chromosomes. When the size of the mini-chromosome was increased, from about 15 kb to about 60 kb, there was a dramatic increase in its mitotic stability in these mutants, but not in the ARS-specific class of mutants. One mutant, mcm17, belonging to the size-dependent class was further characterized. In this mutant, cells carried mini-chromosomes in significantly elevated copy numbers, suggesting a defect in segregation. This defect was largely suppressed in the 60-kb mini-chromosome. A non-centromeric plasmid, the TRP1ARS1 circle, was not affected in its maintenance. This mutant also displayed enhanced chromosome-III loss during mitosis over the wild-type strain, without elevating mitotic recombination. Cloning and sequencing of MCM17 has shown it to be the same as CHL4, a gene required for chromosome stability. This gene is non-essential for growth, as its disruption or deletion from the chromosome did not affect the growth-rate of cells at 23 degrees C or 37 degrees C. This work suggests that centromere-directed segregation of a chromosome in yeast is strongly influenced by its length.
Insights
Certain yeast mini-chromosome-maintenance (mcm) mutants exhibit size-dependent chromosome loss. Increasing mini-chromosome size enhances stability, indicating length influences segregation, particularly in mcm17 mutants.
Area of Science:
- Yeast genetics
- Molecular and cell biology
- Chromosomal instability
Background:
- Mini-chromosome-maintenance (mcm) mutants are known to affect mini-chromosome stability in yeast.
- ARS-specific mcm mutants are characterized by defects in DNA replication initiation.
- Previous studies focused on ARS-specific mutants, leaving other classes less explored.
Purpose of the Study:
- To identify and characterize novel mcm mutants exhibiting size-dependent mini-chromosome loss.
- To investigate the role of chromosome length in mitotic stability and segregation.
- To elucidate the function of the MCM17 gene in maintaining chromosome integrity.
Main Methods:
- Generation and screening of mcm mutants for size-dependent mini-chromosome loss.
- Mitotic stability assays using mini-chromosomes of varying sizes (15 kb to 60 kb).
- Characterization of the mcm17 mutant, including copy number analysis and chromosome loss assays.
- Cloning, sequencing, and functional analysis of the MCM17 gene (CHL4).
Main Results:
- Identified mcm mutants showing increased mini-chromosome stability with larger sizes (up to 60 kb), unlike ARS-specific mutants.
- The mcm17 mutant displayed elevated mini-chromosome copy numbers, indicating a segregation defect suppressed by larger size.
- MCM17 (CHL4) is essential for chromosome stability, but not for cell growth, and its absence leads to increased chromosome III loss.
- The TRP1ARS1 circle plasmid maintenance was unaffected in mcm17 mutants.
Conclusions:
- Chromosome length significantly influences centromere-directed segregation in yeast.
- The MCM17/CHL4 gene plays a crucial role in chromosome segregation, particularly for smaller chromosomes.
- Size-dependent chromosome stability highlights complex regulatory mechanisms in maintaining genome integrity.