Srs2 promotes Mus81-Mms4-mediated resolution of recombination intermediates.
Bottom Line: Second, Srs2 removes Rad51 from DNA to allow access of Mus81-Mms4 to cleave DNA.Concomitantly, Mus81-Mms4 inhibits the helicase activity of Srs2.Taken together, our data point to a coordinated role of Mus81-Mms4 and Srs2 in processing of recombination as well as replication intermediates.
Affiliation: Department of Biology, Masaryk University, Kamenice 5/A7, Brno 625 00, Czech Republic National Centre for Biomolecular Research, Masaryk University, Kamenice 5/A4, Brno 625 00, Czech Republic.Show MeSH
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Mentions: Since the Mus81 protein mediates the interaction with Srs2, we aimed to determine the region of Mus81 protein required for this interaction. We therefore cloned, expressed and purified an N-terminal fragment of Mus81 (V5-His-Mus811–319) and tested its interaction with full-length Srs2. A C-terminal fragment of Mus81 (V5-His-Mus81319–632) was insoluble and could not be used for the analysis. As shown in Figure 2A, the N-terminus of Mus81 was able to retain Srs2 on anti-V5 agarose beads. In addition, we compared the binding between full-length Mus81 and Mus811–319 and found that they both bind the Srs2 (Supplementary Figure S1A). Next, we tested the affinity of Mus811–319 toward various N- and C-terminal truncations of Srs2. Using the GSH-bead pull-down assay, an identical pattern of interaction was observed with full-length Mus81. Similar results were obtained using both Coomassie blue staining and western blot analysis. The inability of the Srs21–700 fragment to interact with Mus811–319 confirms that the interaction domain within Srs2 resides between aa 783–860 (Figure 2B, C and D).
Affiliation: Department of Biology, Masaryk University, Kamenice 5/A7, Brno 625 00, Czech Republic National Centre for Biomolecular Research, Masaryk University, Kamenice 5/A4, Brno 625 00, Czech Republic.