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Cooperativity of Mus81.Mms4 with Rad54 in the resolution of recombination and replication intermediates.

Matulova P, Marini V, Burgess RC, Sisakova A, Kwon Y, Rothstein R, Sung P, Krejci L - J. Biol. Chem. (2009)

Bottom Line: Previous yeast two-hybrid studies have found an interaction of the Mus81 protein with Rad54, a Swi2/Snf2-like factor that serves multiple roles in homologous recombination processes.We propose that Mus81.Mms4 together with Rad54 efficiently process perturbed replication forks to promote recovery and may constitute an alternative mechanism to the resolution/dissolution of the recombination intermediates by Sgs1.Top3.These findings provide functional insights into the biological importance of the higher order complex of Mus81.Mms4 or its orthologue with Rad54.

View Article: PubMed Central - PubMed

Affiliation: National Centre for Biomolecular Research, Masaryk University, Brno 62500, Czech Republic.

ABSTRACT
The Saccharomyces cerevisiae Mus81.Mms4 protein complex, a DNA structure-specific endonuclease, helps preserve genomic integrity by resolving pathological DNA structures that arise from damaged or aborted replication forks and may also play a role in the resolution of DNA intermediates arising through homologous recombination. Previous yeast two-hybrid studies have found an interaction of the Mus81 protein with Rad54, a Swi2/Snf2-like factor that serves multiple roles in homologous recombination processes. However, the functional significance of this novel interaction remains unknown. Here, using highly purified S. cerevisiae proteins, we show that Rad54 strongly stimulates the Mus81.Mms4 nuclease activity on a broad range of DNA substrates. This nuclease enhancement does not require ATP binding nor its hydrolysis by Rad54. We present evidence that Rad54 acts by targeting the Mus81.Mms4 complex to its DNA substrates. In addition, we demonstrate that the Rad54-mediated enhancement of the Mus81.Mms4 (Eme1) nuclease function is evolutionarily conserved. We propose that Mus81.Mms4 together with Rad54 efficiently process perturbed replication forks to promote recovery and may constitute an alternative mechanism to the resolution/dissolution of the recombination intermediates by Sgs1.Top3. These findings provide functional insights into the biological importance of the higher order complex of Mus81.Mms4 or its orthologue with Rad54.

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Physical interaction of Rad54 with Mus81 and Mus81·Mms4. A, purified Rad54 (lane 1), Mus81 (lane 2), Mus81·Mms4 (lane 3), Mus81·Eme1 (lane 4), and Fen1 (lane 5) were resolved by SDS-PAGE and stained with Coomassie Blue. B, Mus81 (4 μg) was mixed with S-protein-agarose in the presence of dsDNA (lane 1–3) or with S-protein-agarose beads coated with Rad54 (4 μg) in the presence of ssDNA (lanes 4–6) or dsDNA (lanes 7–9). The beads were incubated with DNase I (2 units), washed, and treated with SDS to elute bound proteins. The supernatant that contained unbound proteins (S), the wash (W), and the SDS eluate (E) were analyzed by SDS-PAGE. As a positive control, Rad51 (4 μg) was mixed with S-protein-agarose beads coated with Rad54 (4 μg) in the absence of DNA (lanes 10–12) and then analyzed. C, Mus81·Mms4 (3 μg) was mixed with S-protein-agarose beads (lanes 1–3) or S-protein-agarose beads coated with Rad54 (3 μg; lanes 4–6) in the presence of dsDNA. As a negative control, Fen1 (3 μg) was mixed with S-protein-agarose beads coated with Rad54 (3 μg) in the presence of dsDNA (lanes 7–9) and then analyzed. BSA, bovine serum albumin.
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fig1: Physical interaction of Rad54 with Mus81 and Mus81·Mms4. A, purified Rad54 (lane 1), Mus81 (lane 2), Mus81·Mms4 (lane 3), Mus81·Eme1 (lane 4), and Fen1 (lane 5) were resolved by SDS-PAGE and stained with Coomassie Blue. B, Mus81 (4 μg) was mixed with S-protein-agarose in the presence of dsDNA (lane 1–3) or with S-protein-agarose beads coated with Rad54 (4 μg) in the presence of ssDNA (lanes 4–6) or dsDNA (lanes 7–9). The beads were incubated with DNase I (2 units), washed, and treated with SDS to elute bound proteins. The supernatant that contained unbound proteins (S), the wash (W), and the SDS eluate (E) were analyzed by SDS-PAGE. As a positive control, Rad51 (4 μg) was mixed with S-protein-agarose beads coated with Rad54 (4 μg) in the absence of DNA (lanes 10–12) and then analyzed. C, Mus81·Mms4 (3 μg) was mixed with S-protein-agarose beads (lanes 1–3) or S-protein-agarose beads coated with Rad54 (3 μg; lanes 4–6) in the presence of dsDNA. As a negative control, Fen1 (3 μg) was mixed with S-protein-agarose beads coated with Rad54 (3 μg) in the presence of dsDNA (lanes 7–9) and then analyzed. BSA, bovine serum albumin.

Mentions: Affinity Pulldown Studies—S. cerevisiae Rad54-containing protein complexes were captured using S-protein-agarose (Novagen), specific for the S-tag on Rad54. Purified Rad54 (4 μg in Fig. 1B and 3 μg in Fig. 1C) was incubated with Mus81 (4 μg), Mus81·Mms4 (3 μg), Rad51 (4 μg), or Fen1 (3 μg) in 30 μl of buffer T (20 mm Tris-HCl, pH 7.5, 150 mm KCl, 1 mm dithiothreitol, 0.5 mm EDTA, and 0.01% Nonidet P-40) for 30 min at 4 °C. The reactions were mixed with 15 μl of S-Protein-agarose at 4 °C for 30 min and then treated with DNase I (2 units, New England Biolabs) for 10 min at 37 °C. After washing the beads twice with 150 μl of buffer T, the bound proteins were eluted with 30 μl of 5% SDS. The supernatant, wash, and SDS eluate, 10 μl each, were subject to SDS-PAGE analysis.


Cooperativity of Mus81.Mms4 with Rad54 in the resolution of recombination and replication intermediates.

Matulova P, Marini V, Burgess RC, Sisakova A, Kwon Y, Rothstein R, Sung P, Krejci L - J. Biol. Chem. (2009)

Physical interaction of Rad54 with Mus81 and Mus81·Mms4. A, purified Rad54 (lane 1), Mus81 (lane 2), Mus81·Mms4 (lane 3), Mus81·Eme1 (lane 4), and Fen1 (lane 5) were resolved by SDS-PAGE and stained with Coomassie Blue. B, Mus81 (4 μg) was mixed with S-protein-agarose in the presence of dsDNA (lane 1–3) or with S-protein-agarose beads coated with Rad54 (4 μg) in the presence of ssDNA (lanes 4–6) or dsDNA (lanes 7–9). The beads were incubated with DNase I (2 units), washed, and treated with SDS to elute bound proteins. The supernatant that contained unbound proteins (S), the wash (W), and the SDS eluate (E) were analyzed by SDS-PAGE. As a positive control, Rad51 (4 μg) was mixed with S-protein-agarose beads coated with Rad54 (4 μg) in the absence of DNA (lanes 10–12) and then analyzed. C, Mus81·Mms4 (3 μg) was mixed with S-protein-agarose beads (lanes 1–3) or S-protein-agarose beads coated with Rad54 (3 μg; lanes 4–6) in the presence of dsDNA. As a negative control, Fen1 (3 μg) was mixed with S-protein-agarose beads coated with Rad54 (3 μg) in the presence of dsDNA (lanes 7–9) and then analyzed. BSA, bovine serum albumin.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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fig1: Physical interaction of Rad54 with Mus81 and Mus81·Mms4. A, purified Rad54 (lane 1), Mus81 (lane 2), Mus81·Mms4 (lane 3), Mus81·Eme1 (lane 4), and Fen1 (lane 5) were resolved by SDS-PAGE and stained with Coomassie Blue. B, Mus81 (4 μg) was mixed with S-protein-agarose in the presence of dsDNA (lane 1–3) or with S-protein-agarose beads coated with Rad54 (4 μg) in the presence of ssDNA (lanes 4–6) or dsDNA (lanes 7–9). The beads were incubated with DNase I (2 units), washed, and treated with SDS to elute bound proteins. The supernatant that contained unbound proteins (S), the wash (W), and the SDS eluate (E) were analyzed by SDS-PAGE. As a positive control, Rad51 (4 μg) was mixed with S-protein-agarose beads coated with Rad54 (4 μg) in the absence of DNA (lanes 10–12) and then analyzed. C, Mus81·Mms4 (3 μg) was mixed with S-protein-agarose beads (lanes 1–3) or S-protein-agarose beads coated with Rad54 (3 μg; lanes 4–6) in the presence of dsDNA. As a negative control, Fen1 (3 μg) was mixed with S-protein-agarose beads coated with Rad54 (3 μg) in the presence of dsDNA (lanes 7–9) and then analyzed. BSA, bovine serum albumin.
Mentions: Affinity Pulldown Studies—S. cerevisiae Rad54-containing protein complexes were captured using S-protein-agarose (Novagen), specific for the S-tag on Rad54. Purified Rad54 (4 μg in Fig. 1B and 3 μg in Fig. 1C) was incubated with Mus81 (4 μg), Mus81·Mms4 (3 μg), Rad51 (4 μg), or Fen1 (3 μg) in 30 μl of buffer T (20 mm Tris-HCl, pH 7.5, 150 mm KCl, 1 mm dithiothreitol, 0.5 mm EDTA, and 0.01% Nonidet P-40) for 30 min at 4 °C. The reactions were mixed with 15 μl of S-Protein-agarose at 4 °C for 30 min and then treated with DNase I (2 units, New England Biolabs) for 10 min at 37 °C. After washing the beads twice with 150 μl of buffer T, the bound proteins were eluted with 30 μl of 5% SDS. The supernatant, wash, and SDS eluate, 10 μl each, were subject to SDS-PAGE analysis.

Bottom Line: Previous yeast two-hybrid studies have found an interaction of the Mus81 protein with Rad54, a Swi2/Snf2-like factor that serves multiple roles in homologous recombination processes.We propose that Mus81.Mms4 together with Rad54 efficiently process perturbed replication forks to promote recovery and may constitute an alternative mechanism to the resolution/dissolution of the recombination intermediates by Sgs1.Top3.These findings provide functional insights into the biological importance of the higher order complex of Mus81.Mms4 or its orthologue with Rad54.

View Article: PubMed Central - PubMed

Affiliation: National Centre for Biomolecular Research, Masaryk University, Brno 62500, Czech Republic.

ABSTRACT
The Saccharomyces cerevisiae Mus81.Mms4 protein complex, a DNA structure-specific endonuclease, helps preserve genomic integrity by resolving pathological DNA structures that arise from damaged or aborted replication forks and may also play a role in the resolution of DNA intermediates arising through homologous recombination. Previous yeast two-hybrid studies have found an interaction of the Mus81 protein with Rad54, a Swi2/Snf2-like factor that serves multiple roles in homologous recombination processes. However, the functional significance of this novel interaction remains unknown. Here, using highly purified S. cerevisiae proteins, we show that Rad54 strongly stimulates the Mus81.Mms4 nuclease activity on a broad range of DNA substrates. This nuclease enhancement does not require ATP binding nor its hydrolysis by Rad54. We present evidence that Rad54 acts by targeting the Mus81.Mms4 complex to its DNA substrates. In addition, we demonstrate that the Rad54-mediated enhancement of the Mus81.Mms4 (Eme1) nuclease function is evolutionarily conserved. We propose that Mus81.Mms4 together with Rad54 efficiently process perturbed replication forks to promote recovery and may constitute an alternative mechanism to the resolution/dissolution of the recombination intermediates by Sgs1.Top3. These findings provide functional insights into the biological importance of the higher order complex of Mus81.Mms4 or its orthologue with Rad54.

Show MeSH
Related in: MedlinePlus