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Budding yeast mcm10/dna43 mutant requires a novel repair pathway for viability
Yoshio Araki1, Yasuo Kawasaki, Hiroyuki Sasanuma
1Research Institute for Microbial Diseases, Graduate School of Science, Osaka University, 3-1 Yamada-oka, Suita, Osaka, Japan.
Background:
MCM10 is essential for the initiation of chromosomal DNA replication in Saccharomyces cerevisiae. Mcm10p functionally interacts with components of the pre-replicative complex (Mcm2-Mcm7 complex and origin recognition complex) as well as the pre-initiation complex component (Cdc45p) suggesting that it may be a component of the pre-RC as well as the pre-IC. Two-dimensional gel electrophoresis analysis showed that Mcm10p is required not only for the initiation of DNA synthesis at replication origins but also for the smooth passage of replication forks at origins. Genetic analysis showed that MCM10 interacts with components of the elongation machinery such as Pol delta and Pol epsilon, suggesting that it may play a role in elongation replication.
Results:
We show that the mcm10 mutation causes replication fork pausing not only at potentially active origins but also at silent origins. We screened for mutations that are lethal in combination with mcm10-1 and obtained seven mutants named slm1-slm6 for synthetically lethal with mcm10. These mutants comprised six complementation groups that can be divided into three classes. Class 1 includes genes that encode components of the pre-RC and pre-IC and are represented by SLM3, 4 and 5 which are allelic to MCM7, MCM2 and CDC45, respectively. Class 2 includes genes involved in the processing of Okazaki fragments in lagging strand synthesis and is represented by SLM1, which is allelic to DNA2. Class 3 includes novel DNA repair genes represented by SLM2 and SLM6.
Conclusions:
The viability of the mcm10-1 mutant is dependent on a novel repair pathway that may participate either in resolving accumulated replication intermediates or the damage caused by blocked replication forks. These results are consistent with the hypothesis that Mcm10p is required for the passage of replication forks through obstacles such as those created by pre-RCs assembled at active or inactive replication origins.
Insights
The MCM10 protein is crucial for DNA replication initiation and fork progression in yeast. Its absence reveals a novel DNA repair pathway essential for cell survival.
Area of Science:
- Molecular Biology
- Genetics
- Cell Biology
Background:
- MCM10 is vital for initiating DNA replication in Saccharomyces cerevisiae.
- Mcm10p interacts with pre-replicative complex (pre-RC) and pre-initiation complex (pre-IC) proteins.
- Mcm10p is necessary for DNA synthesis initiation and replication fork progression at origins.
Purpose of the Study:
- Investigate the role of MCM10 in DNA replication.
- Identify genes synthetically lethal with mcm10 mutations.
- Elucidate the function of Mcm10p in replication fork dynamics and DNA repair.
Main Methods:
- Screening for synthetically lethal mutations with mcm10-1.
- Genetic analysis of mutant interactions.
- Two-dimensional gel electrophoresis to analyze DNA replication intermediates.
Main Results:
- mcm10 mutations cause replication fork pausing at origins.
- Seven synthetically lethal mutants (slm1-slm6) were identified, falling into three classes.
- Class 1 mutants involved pre-RC/pre-IC components, Class 2 involved Okazaki fragment processing (DNA2), and Class 3 involved novel DNA repair genes.
Conclusions:
- Cell viability in mcm10 mutants depends on a novel repair pathway.
- This pathway likely resolves replication intermediates or damage from blocked forks.
- Mcm10p is required for replication fork passage through obstacles at origins.