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Human Mcm10 regulates the catalytic subunit of DNA polymerase-alpha and prevents DNA damage during replication
Sharbani Chattopadhyay1, Anja-Katrin Bielinsky
1Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA.
Abstract:
In Saccharomyces cerevisiae, minichromosome maintenance protein (Mcm) 10 interacts with DNA polymerase (pol)-alpha and functions as a nuclear chaperone for the catalytic subunit, which is rapidly degraded in the absence of Mcm10. We report here that the interaction between Mcm10 and pol-alpha is conserved in human cells. We used a small interfering RNA-based approach to deplete Mcm10 in HeLa cells, and we observed that the catalytic subunit of pol-alpha, p180, was degraded with similar kinetics as Mcm10, whereas the regulatory pol-alpha subunit, p68, remained unaffected. Simultaneous loss of Mcm10 and p180 inhibited S phase entry and led to an accumulation of already replicating cells in late S/G2 as a result of DNA damage, which triggered apoptosis in a subpopulation of cells. These phenotypes differed considerably from analogous studies in Drosophila embryo cells that did not exhibit a similar arrest. To further dissect the roles of Mcm10 and p180 in human cells, we depleted p180 alone and observed a significant delay in S phase entry and fork progression but little effect on cell viability. These results argue that cells can tolerate low levels of p180 as long as Mcm10 is present to "recycle" it. Thus, human Mcm10 regulates both replication initiation and elongation and maintains genome integrity.
Insights
Human Mcm10 protein is essential for DNA polymerase alpha stability and function, preventing DNA damage and apoptosis. Its loss halts DNA replication, impacting cell cycle progression and genome integrity.
Area of Science:
- Cell Biology
- Molecular Biology
- Genetics
Background:
- Minichromosome maintenance protein (Mcm) 10 acts as a nuclear chaperone for DNA polymerase (pol)-alpha's catalytic subunit in yeast.
- This interaction is crucial for preventing the rapid degradation of the catalytic subunit in the absence of Mcm10.
Purpose of the Study:
- To investigate the conserved interaction between Mcm10 and pol-alpha in human cells.
- To elucidate the roles of Mcm10 and pol-alpha in DNA replication and genome integrity in human cells.
Main Methods:
- Small interfering RNA (siRNA) was used to deplete Mcm10 in HeLa cells.
- The degradation kinetics of pol-alpha subunits (p180 and p68) were analyzed following Mcm10 depletion.
- p180 was also depleted alone to assess its specific effects on cell viability and replication.
Main Results:
- Depletion of Mcm10 in HeLa cells led to the degradation of the catalytic pol-alpha subunit (p180), while the regulatory subunit (p68) remained unaffected.
- Simultaneous loss of Mcm10 and p180 inhibited S phase entry, caused DNA damage, and induced apoptosis.
- Depleting only p180 delayed S phase entry and fork progression but had minimal impact on cell viability when Mcm10 was present.
Conclusions:
- Human Mcm10 is essential for stabilizing pol-alpha and regulating DNA replication initiation and elongation.
- Mcm10 plays a critical role in maintaining genome integrity by preventing DNA damage and apoptosis.
- The presence of Mcm10 allows cells to tolerate lower levels of p180, highlighting a unique regulatory mechanism.
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