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Related Experiment Videos

Pathway correcting DNA replication errors in Saccharomyces cerevisiae

A Morrison1, A L Johnson, L H Johnston

  • 1Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, Research Triangle Park, NC.

The EMBO Journal
|April 1, 1993
PubMed
Summary

Mutations in Saccharomyces cerevisiae POL3 DNA polymerase (delta) and the PMS1 mismatch repair gene increase spontaneous mutation rates. These systems act in series, suggesting a common DNA replication error correction pathway.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • DNA polymerase fidelity is crucial for preventing mutations.
  • Mismatch repair systems correct errors missed by polymerases.
  • Saccharomyces cerevisiae provides a model for studying DNA repair pathways.

Purpose of the Study:

  • To investigate the roles of Saccharomyces cerevisiae POL3 DNA polymerase (delta) exonuclease activity and the PMS1 mismatch repair gene in spontaneous mutation.
  • To determine if these systems function independently or in a coordinated pathway.
  • To compare the identified pathway to known error correction mechanisms in other organisms.

Main Methods:

  • Site-directed mutagenesis of POL3 to create an exonuclease-deficient mutant (pol3-01).
  • Deletion of the PMS1 gene.

Related Experiment Videos

  • Measurement of spontaneous mutation rates at the URA3 reporter gene in haploid and diploid yeast cells.
  • Analysis of cell morphology and growth characteristics.
  • Assessment of PMS1 gene expression and cell cycle periodicity.

    • Main Results:

    • The pol3-01 mutant exhibited a 130-fold increase in mutation rate, while pms1 deletion resulted in a 41-fold increase.
    • The pol3-01 mutant generated most single base mutation classes, indicating broad specificity.
    • Double mutant (pol3-01 pms1) cells showed a multiplicative increase in mutation rate, suggesting a serial pathway.
    • PMS1 transcript levels displayed cell cycle periodicity similar to DNA replication genes, including POL3.

    Conclusions:

    • The POL3 3'-->5' exonuclease and PMS1 mismatch repair system function in a common, serial pathway for DNA replication error correction.
    • This pathway is analogous to the dnaQ-->mutHLS system in Escherichia coli.
    • PMS1 appears to be co-regulated with DNA replication genes, highlighting coordinated genome maintenance.