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Mismatch correction acts as a barrier to homeologous recombination in Saccharomyces cerevisiae
1Department of Biochemistry and Molecular Biology, University of Massachusetts Medical Center, Worcester 01655, USA.
Abstract:
A homeologous mitotic recombination assay was used to test the role of Saccharomyces cerevisiae mismatch repair genes PMS1, MSH2 and MSH3 on recombination fidelity. A homeologous gene pair consisting of S. cerevisiae SPT15 and its S. pombe homolog were present as a direct repeat on chromosome V, with the exogenous S. pombe sequences inserted either upstream or downstream of the endogenous S. cerevisiae gene. Each gene carried a different inactivating mutation, rendering the starting strain Spt15-. Recombinants that regenerated SPT15 function were scored after nonselective growth of the cells. In strains wild type for mismatch repair, homeologous recombination was depressed 150- to 180-fold relative to homologous controls, indicating that recombination between diverged sequences is inhibited. In one orientation of the homeologous gene pair, msh2 or msh3 mutations resulted in 17- and 9.6-fold elevations in recombination and the msh2 msh3 double mutant exhibited an 43-fold increase, implying that each MSH gene can function independently in trans to prevent homeologous recombination. Homologous recombination was not significantly affected by the msh mutations. In the other orientation, only msh2 strains were elevated (12-fold) for homeologous recombination. A mutation in MSH3 did not affect the rate of recombination in this orientation. Surprisingly, a pms1 deletion mutant did not exhibit elevated homeologous recombination.
Insights
Saccharomyces cerevisiae mismatch repair genes MSH2 and MSH3 inhibit homeologous recombination, with mutations in these genes increasing recombination rates. The PMS1 gene did not show a similar effect on recombination fidelity.
Area of Science:
- Molecular Biology
- Genetics
- Yeast Biology
Background:
- Recombination fidelity is crucial for maintaining genome stability.
- Mismatch repair (MMR) pathways play a role in correcting errors during DNA replication and recombination.
- Understanding the role of specific MMR genes in homeologous recombination is essential for comprehending genome evolution and stability.
Purpose of the Study:
- To investigate the function of Saccharomyces cerevisiae mismatch repair genes PMS1, MSH2, and MSH3 in homeologous mitotic recombination.
- To determine the impact of mutations in these MMR genes on recombination fidelity between diverged DNA sequences.
Main Methods:
- Utilized a homeologous mitotic recombination assay in Saccharomyces cerevisiae.
- Employed a direct repeat system on chromosome V involving S. cerevisiae SPT15 and its S. pombe homolog, each with distinct inactivating mutations.
- Scored recombinants that restored SPT15 function after nonselective cell growth.
Main Results:
- Wild-type strains showed a 150- to 180-fold depression of homeologous recombination compared to homologous recombination.
- Mutations in MSH2 or MSH3 independently elevated homeologous recombination (17- and 9.6-fold, respectively), with a 43-fold increase in the msh2 msh3 double mutant.
- MSH2 mutations elevated homeologous recombination 12-fold in one orientation, while MSH3 mutations had no significant effect in that orientation.
- PMS1 deletion mutants did not show elevated homeologous recombination.
Conclusions:
- MSH2 and MSH3 proteins function independently to suppress homeologous recombination in a sequence orientation-dependent manner.
- The MSH2 and MSH3 genes are key players in preventing recombination between diverged DNA sequences.
- The PMS1 gene does not appear to play a significant role in suppressing homeologous recombination in this assay.