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Srs2 promotes Mus81-Mms4-mediated resolution of recombination intermediates.

Chavdarova M, Marini V, Sisakova A, Sedlackova H, Vigasova D, Brill SJ, Lisby M, Krejci L - Nucleic Acids Res. (2015)

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.

View Article: PubMed Central - PubMed

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.

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Mapping of the Srs2 interaction with Mus81. (A) Full-length Srs2 (5 μg) was incubated in the absence or presence of Mus811–319 (5 μg) and anti-V5 agarose beads. Beads were washed, treated with SDS to elute bound proteins and fractions analyzed by Coomassie blue staining. The asterisk shows protein contamination. (B) Srs21–898 (5 μg, lanes 1–4) and Srs21–860 (lanes 5–6) were mixed with Mus811–319 (5 μg) as indicated, followed by incubation with anti-V5 agarose beads. Beads were treated as described above. (C) Srs21–700 (5 μg) was incubated in the absence or presence of Mus811–319 (5 μg) and anti-V5 agarose beads. Beads were analyzed as described above. (D) Mus811–319 (5 μg) was mixed with GSH-beads (lanes 1 and 2) or with GST-Srs2783–1174, -Srs2783–998, -Srs2783–898, -Srs2898–998, or -Srs2998–1174 (5 μg) and GSH-beads (lanes 3–12). The beads were treated as described above. (E) The yeast two-hybrid interaction between Srs2 and Mus81 is mediated by the N-terminus of Mus81. Strain PJ69–4 containing indicated plasmids expressing Srs2 fused to the GAL4 transcription activation domain and N-terminal regions of Mus81 (Mus811–155, Mus811–220 and Mus811–245) fused to the GAL4 DNA-binding domain were spotted as 10-fold serial dilutions on medium lacking tryptophan and leucine (-trp leu) or lacking tryptophan, leucine and histidine (-trp leu his). The empty vector (pGADT7) was included as negative control.
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Figure 2: Mapping of the Srs2 interaction with Mus81. (A) Full-length Srs2 (5 μg) was incubated in the absence or presence of Mus811–319 (5 μg) and anti-V5 agarose beads. Beads were washed, treated with SDS to elute bound proteins and fractions analyzed by Coomassie blue staining. The asterisk shows protein contamination. (B) Srs21–898 (5 μg, lanes 1–4) and Srs21–860 (lanes 5–6) were mixed with Mus811–319 (5 μg) as indicated, followed by incubation with anti-V5 agarose beads. Beads were treated as described above. (C) Srs21–700 (5 μg) was incubated in the absence or presence of Mus811–319 (5 μg) and anti-V5 agarose beads. Beads were analyzed as described above. (D) Mus811–319 (5 μg) was mixed with GSH-beads (lanes 1 and 2) or with GST-Srs2783–1174, -Srs2783–998, -Srs2783–898, -Srs2898–998, or -Srs2998–1174 (5 μg) and GSH-beads (lanes 3–12). The beads were treated as described above. (E) The yeast two-hybrid interaction between Srs2 and Mus81 is mediated by the N-terminus of Mus81. Strain PJ69–4 containing indicated plasmids expressing Srs2 fused to the GAL4 transcription activation domain and N-terminal regions of Mus81 (Mus811–155, Mus811–220 and Mus811–245) fused to the GAL4 DNA-binding domain were spotted as 10-fold serial dilutions on medium lacking tryptophan and leucine (-trp leu) or lacking tryptophan, leucine and histidine (-trp leu his). The empty vector (pGADT7) was included as negative control.

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).


Srs2 promotes Mus81-Mms4-mediated resolution of recombination intermediates.

Chavdarova M, Marini V, Sisakova A, Sedlackova H, Vigasova D, Brill SJ, Lisby M, Krejci L - Nucleic Acids Res. (2015)

Mapping of the Srs2 interaction with Mus81. (A) Full-length Srs2 (5 μg) was incubated in the absence or presence of Mus811–319 (5 μg) and anti-V5 agarose beads. Beads were washed, treated with SDS to elute bound proteins and fractions analyzed by Coomassie blue staining. The asterisk shows protein contamination. (B) Srs21–898 (5 μg, lanes 1–4) and Srs21–860 (lanes 5–6) were mixed with Mus811–319 (5 μg) as indicated, followed by incubation with anti-V5 agarose beads. Beads were treated as described above. (C) Srs21–700 (5 μg) was incubated in the absence or presence of Mus811–319 (5 μg) and anti-V5 agarose beads. Beads were analyzed as described above. (D) Mus811–319 (5 μg) was mixed with GSH-beads (lanes 1 and 2) or with GST-Srs2783–1174, -Srs2783–998, -Srs2783–898, -Srs2898–998, or -Srs2998–1174 (5 μg) and GSH-beads (lanes 3–12). The beads were treated as described above. (E) The yeast two-hybrid interaction between Srs2 and Mus81 is mediated by the N-terminus of Mus81. Strain PJ69–4 containing indicated plasmids expressing Srs2 fused to the GAL4 transcription activation domain and N-terminal regions of Mus81 (Mus811–155, Mus811–220 and Mus811–245) fused to the GAL4 DNA-binding domain were spotted as 10-fold serial dilutions on medium lacking tryptophan and leucine (-trp leu) or lacking tryptophan, leucine and histidine (-trp leu his). The empty vector (pGADT7) was included as negative control.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
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Figure 2: Mapping of the Srs2 interaction with Mus81. (A) Full-length Srs2 (5 μg) was incubated in the absence or presence of Mus811–319 (5 μg) and anti-V5 agarose beads. Beads were washed, treated with SDS to elute bound proteins and fractions analyzed by Coomassie blue staining. The asterisk shows protein contamination. (B) Srs21–898 (5 μg, lanes 1–4) and Srs21–860 (lanes 5–6) were mixed with Mus811–319 (5 μg) as indicated, followed by incubation with anti-V5 agarose beads. Beads were treated as described above. (C) Srs21–700 (5 μg) was incubated in the absence or presence of Mus811–319 (5 μg) and anti-V5 agarose beads. Beads were analyzed as described above. (D) Mus811–319 (5 μg) was mixed with GSH-beads (lanes 1 and 2) or with GST-Srs2783–1174, -Srs2783–998, -Srs2783–898, -Srs2898–998, or -Srs2998–1174 (5 μg) and GSH-beads (lanes 3–12). The beads were treated as described above. (E) The yeast two-hybrid interaction between Srs2 and Mus81 is mediated by the N-terminus of Mus81. Strain PJ69–4 containing indicated plasmids expressing Srs2 fused to the GAL4 transcription activation domain and N-terminal regions of Mus81 (Mus811–155, Mus811–220 and Mus811–245) fused to the GAL4 DNA-binding domain were spotted as 10-fold serial dilutions on medium lacking tryptophan and leucine (-trp leu) or lacking tryptophan, leucine and histidine (-trp leu his). The empty vector (pGADT7) was included as negative control.
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).

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.

View Article: PubMed Central - PubMed

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
Related in: MedlinePlus