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Identification and characterization of MUS81 point mutations that abolish interaction with the SLX4 scaffold protein.

Nair N, Castor D, Macartney T, Rouse J - DNA Repair (Amst.) (2014)

Bottom Line: In this study we looked the other way around by pinpointing amino acid residues in MUS81 that when mutated abolish the interaction with SLX4.These mutations fully rescued the mitomycin C hypersensitivity of MUS81 knockout murine cells, but they were unable to rescue the sensitivity of two different human cell lines defective in MUS81.These data support an SLX4-dependent role for MUS81 in the repair, but not the induction of ICL-induced double-strand breaks.

View Article: PubMed Central - PubMed

Affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, UK.

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MUS81 mutations that cannot interact with SLX4 cause ICL repair defects in human cells but not mouse cells. (A) Mus81−/− MEFs were infected with retroviruses expressing wild-type MUS81, MUS81 24/25AA or MUS81 66/67AA. Wild-type MEFs (WT) and Mus81−/− MEFs infected with empty virus were used as controls. Extracts were subjected to western blotting to test expression (upper panel) or immunoprecipitation with anti-SLX4 (middle panel) and anti-EME1 antibodies (lower panel). (B) Clonogenic survival analysis of Mus81−/− MEFs stably expressing MUS81, MUS81 24/25AA or MUS81 66/67AA, exposed to MMC. For each genotype, cell viability of untreated cells was defined as 100%. Mus81−/− MEFs infected with empty virus were used as controls. Data are represented as mean ± SEM, n = 3. (C) MUS81−/− HCT116 cells were infected with retroviruses expressing MUS81, MUS81 24/25AA or MUS81 66/67AA. Wild-type cells (WT) and MUS81−/− HCT116 cells infected with empty virus were used as controls. Extracts were subjected to western blotting to test expression (upper panel) or immunoprecipitation with anti-MUS81 and anti-SLX4 antibodies (lower panel). (D) Clonogenic survival analysis of MUS81−/− HCT116 cells stably expressing MUS81, MUS81 24/25AA or MUS81 66/67AA, exposed to MMC. For each genotype, cell viability of untreated cells was defined as 100%. Wild-type HCT116 and MUS81−/− HCT116 cells infected with empty virus were used as controls. Data are represented as mean ± SEM, n = 3.
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fig0010: MUS81 mutations that cannot interact with SLX4 cause ICL repair defects in human cells but not mouse cells. (A) Mus81−/− MEFs were infected with retroviruses expressing wild-type MUS81, MUS81 24/25AA or MUS81 66/67AA. Wild-type MEFs (WT) and Mus81−/− MEFs infected with empty virus were used as controls. Extracts were subjected to western blotting to test expression (upper panel) or immunoprecipitation with anti-SLX4 (middle panel) and anti-EME1 antibodies (lower panel). (B) Clonogenic survival analysis of Mus81−/− MEFs stably expressing MUS81, MUS81 24/25AA or MUS81 66/67AA, exposed to MMC. For each genotype, cell viability of untreated cells was defined as 100%. Mus81−/− MEFs infected with empty virus were used as controls. Data are represented as mean ± SEM, n = 3. (C) MUS81−/− HCT116 cells were infected with retroviruses expressing MUS81, MUS81 24/25AA or MUS81 66/67AA. Wild-type cells (WT) and MUS81−/− HCT116 cells infected with empty virus were used as controls. Extracts were subjected to western blotting to test expression (upper panel) or immunoprecipitation with anti-MUS81 and anti-SLX4 antibodies (lower panel). (D) Clonogenic survival analysis of MUS81−/− HCT116 cells stably expressing MUS81, MUS81 24/25AA or MUS81 66/67AA, exposed to MMC. For each genotype, cell viability of untreated cells was defined as 100%. Wild-type HCT116 and MUS81−/− HCT116 cells infected with empty virus were used as controls. Data are represented as mean ± SEM, n = 3.

Mentions: We next checked the ability of MUS81 mutants to rescue MMC hypersensitivity of Mus81−/− MEFs. To this end Mus81−/− MEFs [5] were infected with retroviruses for stable expression of wild-type mouse MUS81 (wt-MUS81) or full-length mouse MUS81 carrying the mutations W24A L25A (MUS81 24/25AA) or L66A Q67A (MUS81 66/67AA). Wild-type and Mus81−/− MEFs were infected with empty viruses to serve as controls. MUS81 expression in the complemented Mus81−/− MEFs was assessed by immunoblotting of whole cell extracts (Fig. 2A, top panel). While wild-type MUS81 and EME1 were detected in SLX4 immunoprecipitates, the MUS81 loss-of-SLX4-interaction mutants i.e. MUS81 24/25AA and MUS81 66/67AA were not (Fig. 2A, middle panel). Similar results were obtained when endogenous EME1 was precipitated from cells complemented with the MUS81 mutants (Fig. 2A, bottom panel). SLX4 was detected in EME1 immunoprecipitates from cells expressing wild-type MUS81, but not MUS81 24/25AA and MUS81 66/67AA mutants. The complemented Mus81−/− MEFs were used in a clonogenic survival assay to test sensitivity to MMC. Mus81−/− MEFs are hypersensitive to MMC compared to wild-type MEFs. This sensitivity was rescued by ectopic expression of wild-type MUS81, and the MUS81 24/25AA and MUS81 66/67AA mutants were indistinguishable from wild-type (Fig. 2B). These data suggest that the role of MUS81 in ICL repair is independent of its interaction with SLX4 at least in murine cells.


Identification and characterization of MUS81 point mutations that abolish interaction with the SLX4 scaffold protein.

Nair N, Castor D, Macartney T, Rouse J - DNA Repair (Amst.) (2014)

MUS81 mutations that cannot interact with SLX4 cause ICL repair defects in human cells but not mouse cells. (A) Mus81−/− MEFs were infected with retroviruses expressing wild-type MUS81, MUS81 24/25AA or MUS81 66/67AA. Wild-type MEFs (WT) and Mus81−/− MEFs infected with empty virus were used as controls. Extracts were subjected to western blotting to test expression (upper panel) or immunoprecipitation with anti-SLX4 (middle panel) and anti-EME1 antibodies (lower panel). (B) Clonogenic survival analysis of Mus81−/− MEFs stably expressing MUS81, MUS81 24/25AA or MUS81 66/67AA, exposed to MMC. For each genotype, cell viability of untreated cells was defined as 100%. Mus81−/− MEFs infected with empty virus were used as controls. Data are represented as mean ± SEM, n = 3. (C) MUS81−/− HCT116 cells were infected with retroviruses expressing MUS81, MUS81 24/25AA or MUS81 66/67AA. Wild-type cells (WT) and MUS81−/− HCT116 cells infected with empty virus were used as controls. Extracts were subjected to western blotting to test expression (upper panel) or immunoprecipitation with anti-MUS81 and anti-SLX4 antibodies (lower panel). (D) Clonogenic survival analysis of MUS81−/− HCT116 cells stably expressing MUS81, MUS81 24/25AA or MUS81 66/67AA, exposed to MMC. For each genotype, cell viability of untreated cells was defined as 100%. Wild-type HCT116 and MUS81−/− HCT116 cells infected with empty virus were used as controls. Data are represented as mean ± SEM, n = 3.
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fig0010: MUS81 mutations that cannot interact with SLX4 cause ICL repair defects in human cells but not mouse cells. (A) Mus81−/− MEFs were infected with retroviruses expressing wild-type MUS81, MUS81 24/25AA or MUS81 66/67AA. Wild-type MEFs (WT) and Mus81−/− MEFs infected with empty virus were used as controls. Extracts were subjected to western blotting to test expression (upper panel) or immunoprecipitation with anti-SLX4 (middle panel) and anti-EME1 antibodies (lower panel). (B) Clonogenic survival analysis of Mus81−/− MEFs stably expressing MUS81, MUS81 24/25AA or MUS81 66/67AA, exposed to MMC. For each genotype, cell viability of untreated cells was defined as 100%. Mus81−/− MEFs infected with empty virus were used as controls. Data are represented as mean ± SEM, n = 3. (C) MUS81−/− HCT116 cells were infected with retroviruses expressing MUS81, MUS81 24/25AA or MUS81 66/67AA. Wild-type cells (WT) and MUS81−/− HCT116 cells infected with empty virus were used as controls. Extracts were subjected to western blotting to test expression (upper panel) or immunoprecipitation with anti-MUS81 and anti-SLX4 antibodies (lower panel). (D) Clonogenic survival analysis of MUS81−/− HCT116 cells stably expressing MUS81, MUS81 24/25AA or MUS81 66/67AA, exposed to MMC. For each genotype, cell viability of untreated cells was defined as 100%. Wild-type HCT116 and MUS81−/− HCT116 cells infected with empty virus were used as controls. Data are represented as mean ± SEM, n = 3.
Mentions: We next checked the ability of MUS81 mutants to rescue MMC hypersensitivity of Mus81−/− MEFs. To this end Mus81−/− MEFs [5] were infected with retroviruses for stable expression of wild-type mouse MUS81 (wt-MUS81) or full-length mouse MUS81 carrying the mutations W24A L25A (MUS81 24/25AA) or L66A Q67A (MUS81 66/67AA). Wild-type and Mus81−/− MEFs were infected with empty viruses to serve as controls. MUS81 expression in the complemented Mus81−/− MEFs was assessed by immunoblotting of whole cell extracts (Fig. 2A, top panel). While wild-type MUS81 and EME1 were detected in SLX4 immunoprecipitates, the MUS81 loss-of-SLX4-interaction mutants i.e. MUS81 24/25AA and MUS81 66/67AA were not (Fig. 2A, middle panel). Similar results were obtained when endogenous EME1 was precipitated from cells complemented with the MUS81 mutants (Fig. 2A, bottom panel). SLX4 was detected in EME1 immunoprecipitates from cells expressing wild-type MUS81, but not MUS81 24/25AA and MUS81 66/67AA mutants. The complemented Mus81−/− MEFs were used in a clonogenic survival assay to test sensitivity to MMC. Mus81−/− MEFs are hypersensitive to MMC compared to wild-type MEFs. This sensitivity was rescued by ectopic expression of wild-type MUS81, and the MUS81 24/25AA and MUS81 66/67AA mutants were indistinguishable from wild-type (Fig. 2B). These data suggest that the role of MUS81 in ICL repair is independent of its interaction with SLX4 at least in murine cells.

Bottom Line: In this study we looked the other way around by pinpointing amino acid residues in MUS81 that when mutated abolish the interaction with SLX4.These mutations fully rescued the mitomycin C hypersensitivity of MUS81 knockout murine cells, but they were unable to rescue the sensitivity of two different human cell lines defective in MUS81.These data support an SLX4-dependent role for MUS81 in the repair, but not the induction of ICL-induced double-strand breaks.

View Article: PubMed Central - PubMed

Affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, UK.

Show MeSH
Related in: MedlinePlus