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The yeast Saccharomyces cerevisiae DNA polymerase IV: possible involvement in double strand break DNA repair
Abstract:
We identified and purified a new DNA polymerase (DNA polymerase IV), which is similar to mammalian DNA polymerase beta, from Saccharomyces cerevisiae and suggested that it is encoded by YCR14C (POLX) on chromosome III. Here, we provided a direct evidence that the purified DNA polymerase IV is indeed encoded by POLX. Strains harboring a pol4 deletion mutation exhibit neither mitotic growth defect nor a meiosis defect, suggesting that DNA polymerase IV participates in nonessential functions in DNA metabolism. The deletion strains did not exhibit UV-sensitivity. However, they did show weak sensitivity to MMS-treatment and exhibited a hyper-recombination phenotype when intragenic recombination was measured during meiosis. Furthermore, MAT alpha pol4 delta segregants had a higher frequency of illegitimate mating with a MAT alpha tester strain than that of wild-type cells. These results suggest that DNA polymerase IV participates in a double-strand break repair pathway. A 3.2kb of the POL4 transcript was weakly expressed in mitotically growing cells. During meiosis, a 2.2 kb POL4 transcript was greatly induced, while the 3.2 kb transcript stayed at constant levels. This induction was delayed in a swi4 delta strain during meiosis, while no effect was observed in a swi6 delta strain.
Insights
DNA polymerase IV, encoded by POLX in yeast, plays a role in DNA repair and recombination. Its deletion causes sensitivity to MMS and increased hyper-recombination during meiosis.
Area of Science:
- Molecular Biology
- Yeast Genetics
- DNA Repair Mechanisms
Background:
- A novel DNA polymerase, DNA polymerase IV, was identified in Saccharomyces cerevisiae.
- This enzyme shares similarities with mammalian DNA polymerase beta.
- Its gene, POLX, was initially suggested to be YCR14C on chromosome III.
Purpose of the Study:
- To provide direct evidence that DNA polymerase IV is encoded by POLX.
- To investigate the function of DNA polymerase IV in yeast DNA metabolism and repair.
- To analyze the expression pattern of the POL4 transcript during different cellular processes.
Main Methods:
- Purification of DNA polymerase IV from Saccharomyces cerevisiae.
- Construction and analysis of pol4 deletion mutant strains.
- Assessment of mitotic and meiotic growth, UV and MMS sensitivity, and recombination frequencies.
- Analysis of illegitimate mating frequencies.
- Transcriptional analysis of POL4 using Northern blotting.
Main Results:
- Direct evidence confirmed that DNA polymerase IV is encoded by POLX.
- Deletion mutants (pol4 delta) showed no mitotic or meiotic growth defects or UV sensitivity.
- pol4 delta strains exhibited weak MMS sensitivity and a hyper-recombination phenotype during meiosis.
- Illegitimate mating frequencies were higher in MAT alpha pol4 delta segregants.
- A 2.2 kb POL4 transcript was induced during meiosis, while a 3.2 kb transcript was weakly expressed in mitotic cells.
- POL4 induction during meiosis was delayed in swi4 delta strains but unaffected in swi6 delta strains.
Conclusions:
- DNA polymerase IV (POLX) is involved in nonessential DNA metabolism functions.
- The enzyme likely participates in a double-strand break repair pathway.
- POL4 expression is regulated during meiosis, with Swi4 potentially involved in its induction.