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Quantitative Proteomics Reveals Dynamic Interactions of the Minichromosome Maintenance Complex (MCM) in the Cellular Response to Etoposide Induced DNA Damage.

Drissi R, Dubois ML, Douziech M, Boisvert FM - Mol. Cell Proteomics (2015)

Bottom Line: The eukaryotic MCM complex consists of six proteins (MCM2-7) that form a heterohexameric ring with DNA helicase activity, which is loaded on chromatin to form the pre-replication complex.Upon entry in S phase, the helicase is activated and opens the DNA duplex to recruit DNA polymerases at the replication fork.The MCM complex thus plays a crucial role during DNA replication, but recent work suggests that MCM proteins could also be involved in DNA repair.

View Article: PubMed Central - PubMed

Affiliation: From the ‡Department of Anatomy and Cell Biology, Université de Sherbrooke, 3201 Jean-Mignault, Sherbrooke, Québec, J1E 4K8, Canada.

No MeSH data available.


Related in: MedlinePlus

Co-localization of MCM2 and ASF1 with sites of DNA damage. U2OS cells grown on glass coverslips were either mock treated (A–D, I–L) or treated with etoposide at 1 μm for 1 h (E–H, M–P) prior to fixation with paraformaldehyde and labeled for immunofluorescence with a γH2AX antibody (B, F, J, N), a MCM2 antibody (C, G) or a ASF1 antibody (K, O). The merge images are shown (D, H, L, and P). Percentage of cells with three or more obvious co-localization foci at different time of recovery following treatment with etoposide for both MCM2 (Q) and ASF1 (R) (more than 100 cells per time point, n = 3).
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Figure 7: Co-localization of MCM2 and ASF1 with sites of DNA damage. U2OS cells grown on glass coverslips were either mock treated (A–D, I–L) or treated with etoposide at 1 μm for 1 h (E–H, M–P) prior to fixation with paraformaldehyde and labeled for immunofluorescence with a γH2AX antibody (B, F, J, N), a MCM2 antibody (C, G) or a ASF1 antibody (K, O). The merge images are shown (D, H, L, and P). Percentage of cells with three or more obvious co-localization foci at different time of recovery following treatment with etoposide for both MCM2 (Q) and ASF1 (R) (more than 100 cells per time point, n = 3).

Mentions: To test whether the interaction between the MCM complex and ASF1 was associated with sites of DNA damage, we assessed the localization of MCM2 and ASF1 in cells before and after DNA damage. DNA damage was induced with etoposide, but with a smaller dose then used in the protein interaction experiments because the signal for γH2AX was too high and undefined in the cells at those higher doses. In the absence of DNA damage, MCM2 and ASF1 were detected throughout the nucleus, but excluded from the nucleolus in the absence of DNA damage (Fig. 7C and 7K), with little to no signal for γH2AX (Fig. 7B and 7J). Following treatment with etoposide for 1 h, a large increase in the signal for γH2AX was observed (Fig. 7F and 7N). Interestingly, we found some partial co-localization of MCM2 with γH2AX (Fig. 7H) in some of the foci, that was present in some of the cells. ASF1 showed an even greater effect on its co-localization with γH2AX following the treatment with etoposide (Fig. 7P). Because not all cells showed a colocalization, we counted the percentage of cells with three or more obvious colocalization foci at different time of recovery following treatment with etoposide for both MCM2 and ASF1 (more than 100 cells per time point, three independent experiments). The number of foci was selected to avoid counting random events. In both cases, we found a significant increase in the colocalization of the protein with γH2AX, confirming the presence of these proteins to sites of DNA damage.


Quantitative Proteomics Reveals Dynamic Interactions of the Minichromosome Maintenance Complex (MCM) in the Cellular Response to Etoposide Induced DNA Damage.

Drissi R, Dubois ML, Douziech M, Boisvert FM - Mol. Cell Proteomics (2015)

Co-localization of MCM2 and ASF1 with sites of DNA damage. U2OS cells grown on glass coverslips were either mock treated (A–D, I–L) or treated with etoposide at 1 μm for 1 h (E–H, M–P) prior to fixation with paraformaldehyde and labeled for immunofluorescence with a γH2AX antibody (B, F, J, N), a MCM2 antibody (C, G) or a ASF1 antibody (K, O). The merge images are shown (D, H, L, and P). Percentage of cells with three or more obvious co-localization foci at different time of recovery following treatment with etoposide for both MCM2 (Q) and ASF1 (R) (more than 100 cells per time point, n = 3).
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Related In: Results  -  Collection

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Figure 7: Co-localization of MCM2 and ASF1 with sites of DNA damage. U2OS cells grown on glass coverslips were either mock treated (A–D, I–L) or treated with etoposide at 1 μm for 1 h (E–H, M–P) prior to fixation with paraformaldehyde and labeled for immunofluorescence with a γH2AX antibody (B, F, J, N), a MCM2 antibody (C, G) or a ASF1 antibody (K, O). The merge images are shown (D, H, L, and P). Percentage of cells with three or more obvious co-localization foci at different time of recovery following treatment with etoposide for both MCM2 (Q) and ASF1 (R) (more than 100 cells per time point, n = 3).
Mentions: To test whether the interaction between the MCM complex and ASF1 was associated with sites of DNA damage, we assessed the localization of MCM2 and ASF1 in cells before and after DNA damage. DNA damage was induced with etoposide, but with a smaller dose then used in the protein interaction experiments because the signal for γH2AX was too high and undefined in the cells at those higher doses. In the absence of DNA damage, MCM2 and ASF1 were detected throughout the nucleus, but excluded from the nucleolus in the absence of DNA damage (Fig. 7C and 7K), with little to no signal for γH2AX (Fig. 7B and 7J). Following treatment with etoposide for 1 h, a large increase in the signal for γH2AX was observed (Fig. 7F and 7N). Interestingly, we found some partial co-localization of MCM2 with γH2AX (Fig. 7H) in some of the foci, that was present in some of the cells. ASF1 showed an even greater effect on its co-localization with γH2AX following the treatment with etoposide (Fig. 7P). Because not all cells showed a colocalization, we counted the percentage of cells with three or more obvious colocalization foci at different time of recovery following treatment with etoposide for both MCM2 and ASF1 (more than 100 cells per time point, three independent experiments). The number of foci was selected to avoid counting random events. In both cases, we found a significant increase in the colocalization of the protein with γH2AX, confirming the presence of these proteins to sites of DNA damage.

Bottom Line: The eukaryotic MCM complex consists of six proteins (MCM2-7) that form a heterohexameric ring with DNA helicase activity, which is loaded on chromatin to form the pre-replication complex.Upon entry in S phase, the helicase is activated and opens the DNA duplex to recruit DNA polymerases at the replication fork.The MCM complex thus plays a crucial role during DNA replication, but recent work suggests that MCM proteins could also be involved in DNA repair.

View Article: PubMed Central - PubMed

Affiliation: From the ‡Department of Anatomy and Cell Biology, Université de Sherbrooke, 3201 Jean-Mignault, Sherbrooke, Québec, J1E 4K8, Canada.

No MeSH data available.


Related in: MedlinePlus