Human Mcm10 regulates the catalytic subunit of DNA polymerase-alpha and prevents DNA damage during replication.
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.
Chattopadhyay
Sharbani
S
Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA.
Bielinsky
Anja-Katrin
AK
D017209:Apoptosis; D020134:Catalytic Domain; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002457:Cell Extracts; D049109:Cell Proliferation; D002470:Cell Survival; D064447:Checkpoint Kinase 2; D004249:DNA Damage; D004256:DNA Polymerase I / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication; D015536:Down-Regulation / Q000235:genetics; D004789:Enzyme Activation; D006367:HeLa Cells; D006657:Histones / Q000378:metabolism; D006801:Humans; D064110:Minichromosome Maintenance Proteins; D008957:Models, Genetic; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D016196:S Phase; D013816:Thermodynamics
D018797:Cell Cycle Proteins; D002457:Cell Extracts; C468783:H2AX protein, human; D006657:Histones; C489613:MCM10 protein, human; D034741:RNA, Small Interfering; D064447:Checkpoint Kinase 2; C578634:CHEK2 protein, human; D017346:Protein Serine-Threonine Kinases; D004256:DNA Polymerase I; D064110:Minichromosome Maintenance Proteins
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