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Mapping and characterizing a new DNA replication mutant in Saccharomyces cerevisiae
S L Eberly1, A Sakai, A Sugino
1Laboratory of Genetics, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709.
Yeast (Chichester, England)
|March 1, 1989
Summary
The mak1-3 mutation in Saccharomyces cerevisiae is a novel DNA replication mutation, distinct from TOP1, and is essential for DNA synthesis initiation.
Area of Science:
- Molecular Biology
- Genetics
- Yeast Biology
Background:
- The mak1-3 mutation in Saccharomyces cerevisiae confers temperature-sensitive growth.
- Previous mak1 mutations (mak1-1, 1-2, 1-4) were identified as DNA topoisomerase I (TOP1) deficient.
- The chromosomal location of MAK1-3 was distinct from TOP1.
Purpose of the Study:
- To characterize the mak1-3 mutation in Saccharomyces cerevisiae.
- To determine the specific role of the MAK1 gene product in DNA replication.
- To re-evaluate the classification of the mak1-3 mutation.
Main Methods:
- Genetic background modification for detailed characterization.
- In vivo macromolecular synthesis analysis at restrictive temperatures.
- In vitro DNA synthesis assays using synchronized cell populations and plasmid pARS1.
Main Results:
- The mak1-3 mutant is deficient in DNA replication initiation at the restrictive temperature.
- Crude extracts from mak1-3 mutant cells showed temperature-sensitive in vitro DNA synthesis.
- The mak1-3 mutation complements all examined DNA synthesis mutants and maps to a new locus.
Conclusions:
- The mak1-3 mutation represents a newly identified DNA replication mutation.
- The MAK1 gene product is directly involved in the initiation of DNA replication.
- The mak1-3 mutation has been renamed 'dna1' due to its distinct characteristics and function.