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DNA polymerase III is required for DNA repair in Saccharomyces cerevisiae
W Suszek1, H Baranowska, J Zuk
1Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw.
Abstract:
We have studied the role of DNA polymerase III, encoded in S. cerevisiae by the CDC2 gene, in the repair of yeast nuclear DNA. It was found that the repair of MMS-induced single-strand breaks is defective in the DNA polymerase III temperature-sensitive mutant cdc2-1 at the restrictive temperature (37 degrees C), but is not affected at the permissive temperature (23 degrees C). Under conditions where only a small number of lesions was introduced into DNA (80% survival), the repair of MMS-induced damage could also be observed in the mutant at the restrictive temperature, although with low efficiency. When the quantity of lesions increased (50% survival or less), the repair of single-strand breaks was blocked. At the same time we observed a high rate of reversion in the meth, his and trp loci of the cdc2-1 mutant under restrictive conditions. The results presented suggest that DNA polymerase III is involved in the repair of MMS-induced lesions in yeast DNA and that the cdc2-1 mutation affects the proofreading activity of this polymerase.
Insights
DNA polymerase III, encoded by the CDC2 gene in yeast, is crucial for repairing DNA damage. A mutation in this gene impairs the repair of MMS-induced DNA breaks, particularly at higher temperatures.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- DNA polymerase III plays a vital role in DNA replication and repair.
- The CDC2 gene in Saccharomyces cerevisiae encodes DNA polymerase III.
- DNA repair mechanisms are essential for maintaining genomic integrity.
Purpose of the Study:
- To investigate the role of DNA polymerase III in the repair of methyl methanesulfonate (MMS)-induced DNA damage in yeast.
- To determine the effect of a specific temperature-sensitive mutation (cdc2-1) in DNA polymerase III on DNA repair efficiency.
Main Methods:
- Utilized a temperature-sensitive mutant of Saccharomyces cerevisiae (cdc2-1) defective in DNA polymerase III.
- Exposed yeast cells to methyl methanesulfonate (MMS) to induce single-strand DNA breaks.
- Assessed DNA repair efficiency at permissive (23°C) and restrictive (37°C) temperatures.
- Monitored reversion rates at specific genetic loci (meth, his, trp) as an indicator of DNA damage and repair.
Main Results:
- The cdc2-1 mutant showed defective repair of MMS-induced single-strand breaks at the restrictive temperature (37°C).
- DNA repair occurred with low efficiency in the mutant at 37°C when DNA damage was minimal (80% survival).
- Repair of single-strand breaks was completely blocked in the mutant at 37°C under conditions of higher DNA damage (≤50% survival).
- A high rate of reversion was observed in the meth, his, and trp loci of the cdc2-1 mutant at the restrictive temperature.
Conclusions:
- DNA polymerase III is involved in the repair of MMS-induced DNA lesions in yeast.
- The cdc2-1 mutation likely impairs the proofreading activity of DNA polymerase III, affecting its DNA repair function.
- The study highlights the importance of DNA polymerase III's proofreading function in maintaining genomic stability against alkylating agents like MMS.