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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

Generation of inducible stable cell lines expressing GFP-MCM2 and GFP-MCM5.A, and B, GPF-MCM2 and GFP-MCM5 were generated by Flp-In recombination into U2OS cells at a specific integration site. Whole cell extracts from either non-induced U2OS cells (A and B, lane 1 and 3), or from cells induced for expression of GFP-MCM2 (A, lane 2 and 4) or GFP-MCM5 (B, lane 2 and 4) were separated by SDS-PAGE and immunoblotted with a GFP antibody (A and B, lane 1–2), with a MCM2 antibody (A, lane 3–4) and with a MCM5 antibody (B, lane 3–4) to confirm expression of the GFP-tagged proteins. C–F) Cells induced for expression of GFP-MCM2 (C–D) and GFP-MCM5 (E–F) were fixed and labeled for immunofluorescence using a GFP antibody (D and F) and cells were visualized by phase contrast (C–E). Scale bar = 30 μm.
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Figure 2: Generation of inducible stable cell lines expressing GFP-MCM2 and GFP-MCM5.A, and B, GPF-MCM2 and GFP-MCM5 were generated by Flp-In recombination into U2OS cells at a specific integration site. Whole cell extracts from either non-induced U2OS cells (A and B, lane 1 and 3), or from cells induced for expression of GFP-MCM2 (A, lane 2 and 4) or GFP-MCM5 (B, lane 2 and 4) were separated by SDS-PAGE and immunoblotted with a GFP antibody (A and B, lane 1–2), with a MCM2 antibody (A, lane 3–4) and with a MCM5 antibody (B, lane 3–4) to confirm expression of the GFP-tagged proteins. C–F) Cells induced for expression of GFP-MCM2 (C–D) and GFP-MCM5 (E–F) were fixed and labeled for immunofluorescence using a GFP antibody (D and F) and cells were visualized by phase contrast (C–E). Scale bar = 30 μm.

Mentions: To further characterize the involvement of the MCM complex in the cellular response to DNA damage, we decided to identify proteins interacting with different MCM proteins in the absence and presence of DNA damage. First, we generated inducible stable cell lines expressing MCM2 and MCM5 at close to endogenous levels. These cell lines were generated using LAP1-tagged wild type MCM2 and MCM5 with a GFP at the N-terminal (Fig. 2) by flp-in recombination into U2OS cells that have a single integration site. The exogenous GFP-MCM2 and GFP-MCM5 proteins were found in similar levels compared with the endogenous proteins (Fig. 2A and 2B, lanes 4). In fact, expression of the exogenous protein appear to reduce the amount of the endogenous protein expressed, suggesting a possible mechanism regulating the total amount of MCM proteins present in the cell (Fig. 2A and 2B, compare lanes 3 and 4 and supplemental Fig. 2). The GFP-tagged proteins is mostly nuclear as demonstrated by immunofluorescence microscopy (Fig. 2C and 2D).


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)

Generation of inducible stable cell lines expressing GFP-MCM2 and GFP-MCM5.A, and B, GPF-MCM2 and GFP-MCM5 were generated by Flp-In recombination into U2OS cells at a specific integration site. Whole cell extracts from either non-induced U2OS cells (A and B, lane 1 and 3), or from cells induced for expression of GFP-MCM2 (A, lane 2 and 4) or GFP-MCM5 (B, lane 2 and 4) were separated by SDS-PAGE and immunoblotted with a GFP antibody (A and B, lane 1–2), with a MCM2 antibody (A, lane 3–4) and with a MCM5 antibody (B, lane 3–4) to confirm expression of the GFP-tagged proteins. C–F) Cells induced for expression of GFP-MCM2 (C–D) and GFP-MCM5 (E–F) were fixed and labeled for immunofluorescence using a GFP antibody (D and F) and cells were visualized by phase contrast (C–E). Scale bar = 30 μm.
© Copyright Policy - open-access
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Figure 2: Generation of inducible stable cell lines expressing GFP-MCM2 and GFP-MCM5.A, and B, GPF-MCM2 and GFP-MCM5 were generated by Flp-In recombination into U2OS cells at a specific integration site. Whole cell extracts from either non-induced U2OS cells (A and B, lane 1 and 3), or from cells induced for expression of GFP-MCM2 (A, lane 2 and 4) or GFP-MCM5 (B, lane 2 and 4) were separated by SDS-PAGE and immunoblotted with a GFP antibody (A and B, lane 1–2), with a MCM2 antibody (A, lane 3–4) and with a MCM5 antibody (B, lane 3–4) to confirm expression of the GFP-tagged proteins. C–F) Cells induced for expression of GFP-MCM2 (C–D) and GFP-MCM5 (E–F) were fixed and labeled for immunofluorescence using a GFP antibody (D and F) and cells were visualized by phase contrast (C–E). Scale bar = 30 μm.
Mentions: To further characterize the involvement of the MCM complex in the cellular response to DNA damage, we decided to identify proteins interacting with different MCM proteins in the absence and presence of DNA damage. First, we generated inducible stable cell lines expressing MCM2 and MCM5 at close to endogenous levels. These cell lines were generated using LAP1-tagged wild type MCM2 and MCM5 with a GFP at the N-terminal (Fig. 2) by flp-in recombination into U2OS cells that have a single integration site. The exogenous GFP-MCM2 and GFP-MCM5 proteins were found in similar levels compared with the endogenous proteins (Fig. 2A and 2B, lanes 4). In fact, expression of the exogenous protein appear to reduce the amount of the endogenous protein expressed, suggesting a possible mechanism regulating the total amount of MCM proteins present in the cell (Fig. 2A and 2B, compare lanes 3 and 4 and supplemental Fig. 2). The GFP-tagged proteins is mostly nuclear as demonstrated by immunofluorescence microscopy (Fig. 2C and 2D).

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