Related Experiment Videos
Characterization of insertion mutations in the Saccharomyces cerevisiae MSH1 and MSH2 genes: evidence for separate
1Division of Cellular and Molecular Biology, Dana-Farber Cancer Institute, Boston, Massachusetts 02115.
Abstract:
The MSH1 and MSH2 genes of Saccharomyces cerevisiae are predicted to encode proteins that are homologous to the Escherichia coli MutS and Streptococcus pneumoniae HexA proteins and their homologs. Disruption of the MSH1 gene caused a petite phenotype which was established rapidly. A functional MSH1 gene present on a single-copy centromere plasmid was incapable of rescuing the established msh1 petite phenotype. Analysis of msh1 strains demonstrated that mutagenesis and large-scale rearrangement of mitochondrial DNA had occurred. 4',6-Diamidino-2-phenylindole (DAPI) staining of msh1 yeast revealed an aberrant distribution of mtDNA. Haploid msh2 mutants displayed an increase of 85-fold in the rate of spontaneous mutation to canavanine resistance. Sporulation of homozygous msh2/msh2 diploids gave rise to a high level of lethality which was compounded during increased vegetative growth prior to sporulation. msh2 mutations also affected gene conversion of two HIS4 alleles. The his4x mutation, lying near the 5' end of the gene, was converted with equal frequency in both wild-type and msh2 strains. However, many of the events in the msh2 background were post-meiotic segregation (PMS) events (46.4%) while none (< 0.25%) of the aberrant segregations in wild type were PMS events. The his4b allele, lying 1.6 kb downstream of his4x, was converted at a 10-fold higher frequency in the msh2 background than in the corresponding wild-type strain. Like the his4x allele, his4b showed a high level of PMS (30%) in the msh2 background compared to the corresponding wild-type strain where no (< 0.26%) PMS events were observed. These results indicate that MSH1 plays a role in repair or stability of mtDNA and MSH2 plays a role in repair of 4-bp insertion/deletion mispairs in the nucleus.
Insights
The MSH1 gene is crucial for mitochondrial DNA stability in yeast, while MSH2 repairs nuclear DNA mismatches. Disrupting MSH1 causes mitochondrial defects, and MSH2 mutations lead to increased nuclear mutations and aberrant segregation.
Area of Science:
- Molecular Biology
- Genetics
- Yeast Biology
Background:
- The MSH1 and MSH2 genes in Saccharomyces cerevisiae encode proteins homologous to bacterial DNA repair enzymes.
- Understanding the functions of these genes is vital for comprehending DNA repair mechanisms in eukaryotes.
Purpose of the Study:
- To investigate the roles of MSH1 and MSH2 in DNA repair and genome stability in yeast.
- To characterize the phenotypes associated with disruptions in MSH1 and MSH2.
Main Methods:
- Gene disruption of MSH1 and MSH2 in Saccharomyces cerevisiae.
- Phenotypic analysis including petite phenotype assessment, mitochondrial DNA (mtDNA) distribution using DAPI staining, and mutation rate determination.
- Analysis of gene conversion and post-meiotic segregation (PMS) events for HIS4 alleles.
Main Results:
- MSH1 disruption resulted in a petite phenotype, mtDNA mutagenesis, large-scale rearrangement, and aberrant mtDNA distribution.
- MSH2 mutants exhibited an 85-fold increase in spontaneous mutation rates to canavanine resistance.
- MSH2 mutations led to increased post-meiotic segregation events and altered gene conversion frequencies at the HIS4 locus.
Conclusions:
- MSH1 is essential for the stability and integrity of mitochondrial DNA in yeast.
- MSH2 plays a significant role in the repair of 4-base pair insertion/deletion mismatches within the nuclear genome.
- These findings highlight distinct roles for MSH1 and MSH2 in maintaining genome stability in different cellular compartments.