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DNA polymerase of Ustilago maydis: partial characterization of the enzyme and a pol 1 mutation
Abstract:
The major DNA polymerase activity of wild-type U. maydis has been extensively purified. It possesses a molecular weight of about 150,000 daltons and appears to require a DNA primer with a 3'-hydroxyl terminus as well as a template. The polymerase activity has also been purified from the pol 1-1 strain, which is temperature sensitive fro growth and DNA synthesis, and which at the restrictive temperature contains only 10-25% levels of the DNA polymerase activity obtained from wild-type strains. It was similar in all properties studied, except that the activity was thermolabile at 40 degrees C compared to that from the wild-type strain. Physiological studies on the mutant showed that it was only slightly sensitive to UV, ionising radiation and nitrosoguanidine at the permissive temperature, and was proficient in genetic recombination. The results suggest that the pol 1-1 gene product does not play an important role in repair and recombination processes within the cell, and that its primary function lies in replication.
Insights
Researchers purified DNA polymerase from U. maydis, finding the pol 1-1 mutant enzyme is thermolabile. This suggests the pol 1-1 gene product is crucial for DNA replication, not repair or recombination.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- DNA polymerases are essential enzymes for DNA replication, repair, and recombination.
- Ustilago maydis (U. maydis) is a model organism for studying fungal genetics and molecular biology.
- Mutations in DNA polymerase genes can lead to temperature-sensitive phenotypes affecting DNA synthesis.
Purpose of the Study:
- To characterize the major DNA polymerase activity in wild-type U. maydis.
- To investigate the properties of DNA polymerase from the temperature-sensitive pol 1-1 mutant strain.
- To determine the role of the pol 1-1 gene product in U. maydis DNA metabolism.
Main Methods:
- Extensive purification of DNA polymerase activity from wild-type and pol 1-1 U. maydis strains.
- Biochemical characterization of purified DNA polymerase, including molecular weight determination and primer/template requirements.
- Physiological studies on the pol 1-1 mutant, assessing sensitivity to DNA-damaging agents and proficiency in genetic recombination.
Main Results:
- Wild-type U. maydis DNA polymerase has a molecular weight of approximately 150,000 daltons and requires a DNA primer and template.
- The pol 1-1 mutant exhibits significantly reduced DNA polymerase activity (10-25%) at restrictive temperatures and its polymerase is thermolabile.
- The pol 1-1 mutant shows only slight sensitivity to UV, ionizing radiation, and nitrosoguanidine, and is proficient in genetic recombination.
Conclusions:
- The pol 1-1 gene product is likely the major DNA polymerase in U. maydis.
- The thermolability of the mutant polymerase indicates its critical role in DNA replication.
- The pol 1-1 gene product does not appear to be essential for DNA repair or recombination processes.