Identification and characterization of MUS81 point mutations that abolish interaction with the SLX4 scaffold protein.
Bottom Line: It was previously reported that mutations in SLX4 that abolished interaction with MUS81 affected ICL repair in human cells but not in murine cells.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.
Affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, UK.Show MeSH
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Mentions: We next checked the ability of MUS81 mutants to rescue MMC hypersensitivity of Mus81−/− MEFs. To this end Mus81−/− MEFs  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.
Affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, UK.