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Two mcm3 mutations affect different steps in the initiation of DNA replication
Ming Lei1, Irene H Cheng, Louis A Roberts
1Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, WI 53226, USA. mlei@mcw.edu
Abstract:
Mcm3 is a subunit of the hexameric MCM2-7 complex required for the initiation and elongation of DNA replication in eukaryotes. We have characterized two mutant alleles, mcm3-1 and mcm3-10, in Saccharomyces cerevisiae and showed that they are defective at different steps of the replication initiation process. Mcm3-10 contains a P118L substitution that compromises its interaction with Mcm5 and the recruitment of Mcm3 and Mcm7 to a replication origin. P118 is conserved between Mcm3, Mcm4, Mcm5, and Mcm7. An identical substitution of this conserved residue in Mcm5 (P83L of mcm5-bob1) strengthens the interaction between Mcm3 and Mcm5 and allows cells to enter S phase independent of Cdc7-Dbf4 kinase (Hardy, C. F., Dryga, O., Pahl, P. M. B., and Sclafani, R. A. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 3151-3155). Mcm3-1 contains a G246E mutation that diminishes the efficiency of replication initiation (Yan, H., Merchant, A. M., and Tye, B. K. (1993) Genes Dev. 7, 2149-2160) but not its interaction with Mcm5 or recruitment of the MCM2-7 complex to replication origin. These observations indicate that Mcm3-10 is defective in a step before, and Mcm3-1 is defective in a step after the recruitment of the MCM2-7 complex to replication origins.
Insights
Two DNA replication mutants in yeast, mcm3-1 and mcm3-10, reveal distinct defects in replication initiation. Mcm3-10 impairs MCM complex recruitment, while Mcm3-1 affects initiation efficiency post-recruitment.
Area of Science:
- Molecular Biology
- Genetics
- Cell Biology
Background:
- The MCM2-7 complex is essential for eukaryotic DNA replication initiation and elongation.
- Mcm3 is a critical subunit of the MCM2-7 complex.
- Understanding Mcm3 function is key to deciphering replication control.
Purpose of the Study:
- To characterize two Saccharomyces cerevisiae Mcm3 mutant alleles, mcm3-1 and mcm3-10.
- To determine the specific defects in DNA replication initiation caused by these mutations.
Main Methods:
- Genetic analysis of mcm3-1 and mcm3-10 mutant alleles in yeast.
- Biochemical assays to assess Mcm3 interactions and MCM complex recruitment to origins.
- Comparison with previously characterized mutations in Mcm3 and Mcm5.
Main Results:
- Mcm3-10 mutation (P118L) disrupts Mcm3-Mcm5 interaction and MCM complex recruitment to replication origins.
- Mcm3-1 mutation (G246E) reduces replication initiation efficiency but does not affect MCM complex recruitment.
- The P118 residue is conserved and crucial for Mcm3 function.
Conclusions:
- Mcm3-10 is defective in an early step of replication initiation, prior to MCM complex recruitment.
- Mcm3-1 is defective in a later step of replication initiation, after MCM complex recruitment.
- These findings highlight distinct roles for Mcm3 in the temporal regulation of DNA replication initiation.