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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.
Abstract:
Mutation of predicted 3'-->5' exonuclease active site residues of Saccharomyces cerevisiae POL3 DNA polymerase (delta) or deletion of the PMS1 mismatch repair gene lead to relative (to wild type) spontaneous mutation rates of approximately 130 and 41, respectively, measured at a URA3 reporter gene inserted near to a defined replication origin. The POL3 exonuclease-deficient mutant pol3-01 generated most classes of single base mutation in URA3, indicating a broad specificity that generally corresponds to that of the PMS1 system. pol3-01 pms1 haploid cells ceased growth after a few divisions with no unique terminal cell morphology. A pol3-01/pol3-01 pms1/pms1 diploid was viable and displayed an estimated URA3 relative mutation rate of 2 x 10(4), which we calculate to be catastrophically high in a haploid. The relationship between the relative mutation rates of pol3-01 and pms1 was multiplicative, indicating action in series. The PMS1 transcript showed the same cell cycle periodicity as those of a set of DNA replication genes that includes POL3, suggesting PMS1 is co-regulated with these genes. We propose that the POL3 3'-->5' exonuclease and the PMS1 mismatch repair system act on a common pathway analogous to the dnaQ-->mutHLS pathway of DNA replication error correction in Escherichia coli.
Insights
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.
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.
- 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.