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Mcm2 phosphorylation and the response to replicative stress
Brent E Stead1, Christopher J Brandl, Matthew K Sandre
1Department of Biochemistry, Schulich School of Medicine & Dentistry, University of Western Ontario, London, ON, N6A 5C1, Canada.
BMC Genetics
|May 9, 2012
Summary
Dbf4-dependent kinase Cdc7 (DDK) phosphorylation of Mcm2 is crucial for the DNA damage response. Loss of these sites impairs cell proliferation and increases mutation rates, highlighting DDK
Area of Science:
- Cell cycle regulation
- DNA replication
- DNA repair
Background:
- The minichromosome maintenance (Mcm)2-7 complex functions as the replicative helicase in eukaryotes.
- Dbf4-dependent kinase Cdc7 (DDK) phosphorylates Mcm2, a key regulator of cell proliferation and response to replicative stress.
- Previous studies indicated Mcm2 phosphorylation sites are essential for responding to caffeine and methyl methanesulfonate (MMS).
Purpose of the Study:
- To investigate the role of DDK-mediated phosphorylation of Mcm2 in response to various genotoxic agents and DNA damage.
- To identify genes that interact synthetically lethally or suppress the phenotypes associated with loss of Mcm2 phosphorylation sites.
Main Methods:
- Created a Saccharomyces cerevisiae strain with alanine mutations at DDK phosphorylation sites (S164 and S170) in Mcm2 (mcm2AA).
- Assessed sensitivity of mcm2AA strains to hydroxyurea (HU), 5-fluorouracil (5-FU), and phleomycin.
- Performed genome-wide screens to identify synthetic lethal interactions and suppressors of mcm2AA phenotypes.
- Measured spontaneous mutation rates using CAN1 forward mutation assay.
Main Results:
- The mcm2AA strain exhibited sensitivity to HU and 5-FU, but not phleomycin.
- Synthetic lethal screens identified genes involved in genome integrity and oxidative stress.
- The mcm2AA strain showed an increased spontaneous mutation rate, while a phosphomimetic mcm2EE strain showed a decreased rate.
- Suppressor screens identified genes that decrease DNA damage, enhance homologous recombination, or slow replication forks.
Conclusions:
- DDK-mediated phosphorylation of Mcm2 is essential for the cellular response to replicative stress and certain types of DNA damage.
- Phosphorylation of Mcm2 likely modulates Mcm2-7 helicase activity, stabilizing replication forks under stress conditions.
- This phosphorylation event plays a critical role in maintaining genome stability.
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