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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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Rad54 concentration-dependent enhancement of Mus81·Mms4-mediated 3′ DNA flap cleavage. Reaction mixtures containing 3′ DNA flap (6 nm), Mus81·Mms4 (0.25 nm), and the indicated amount of Rad54 (0.5, 1, 2, 4, 8, or 16 nm) were incubated at 37 °C for 10 min (A) and 30 min (B) and then analyzed. C, quantification of the data in A and B with S.D. based on three independent experiments. D, plot of percent of product formation as a logarithm of Rad54 concentration with indicated median effective concentration (EC50).
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fig2: Rad54 concentration-dependent enhancement of Mus81·Mms4-mediated 3′ DNA flap cleavage. Reaction mixtures containing 3′ DNA flap (6 nm), Mus81·Mms4 (0.25 nm), and the indicated amount of Rad54 (0.5, 1, 2, 4, 8, or 16 nm) were incubated at 37 °C for 10 min (A) and 30 min (B) and then analyzed. C, quantification of the data in A and B with S.D. based on three independent experiments. D, plot of percent of product formation as a logarithm of Rad54 concentration with indicated median effective concentration (EC50).

Mentions: Rad54 Stimulates Cleavage of 3′ DNA Flap—The physical interaction between Rad54 and Mus81 together with the demonstrated binding preference of Rad54 for substrates that Mus81·Mms4 acts on prompted us to test whether Rad54 might modulate the nuclease activity of the Mus81·Mms4 complex. We first used the 3′ DNA flap substrate, as it is bound by Rad54 with higher affinity and represents a relevant DNA intermediate that arises during DNA repair or replication. We performed a Rad54 protein titration with 0.25 nm Mus81·Mms4, an amount that could cleave only a small fraction of the substrate (supplemental Fig. 2, A and C). Importantly, strong enhancement of the Mus81·Mms4 DNA cleavage activity occurred in a Rad54 protein concentration-dependent manner. Flap cleavage was stimulated 3-fold by 1 nm Rad54 and 5-fold by 2 nm Rad54, and complete incision of the substrate was seen when 8 nm Rad54 was added (Fig. 2, A–C). The plot of the percent product formed as a logarithmic function of Rad54 concentration yields a sigmoid curve for stimulation, with the median effective concentration EC50 = 1.56 nm (Fig. 2D). This Rad54 amount corresponds to 6 times that of Mus81·Mms4 heterodimer. Because Rad54 forms oligomeric complexes on DNA (22, 29, 30), our results are consistent with the premise that optimal stimulation of Mus81·Mms4 occurs upon assembly of a Rad54 oligomer. As expected, Rad54 alone was devoid of nucleolytic activity (Fig. 2, A and B, lane 9). Time course experiments provided further details on the effect of Rad54 on the rate of Mus81·Mms4-mediated DNA cleavage (Fig. 3). We note that the presence of Rad54 does not affect the position of the cleavage site within the flap structure (data not shown). In addition, the presence of His6 affinity tag on Mms4 does not have any significant effect on the nuclease activity of Mus81·Mms4 or on the enhancement of substrate cleavage by Rad54 (supplemental Fig. 3). Taken together, the results demonstrate that Rad54 can strongly stimulate the ability of Mus81·Mms4 to cleave the 3′ DNA flap structure and also suggest that optimal enhancement is contingent upon oligomerization of Rad54.


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)

Rad54 concentration-dependent enhancement of Mus81·Mms4-mediated 3′ DNA flap cleavage. Reaction mixtures containing 3′ DNA flap (6 nm), Mus81·Mms4 (0.25 nm), and the indicated amount of Rad54 (0.5, 1, 2, 4, 8, or 16 nm) were incubated at 37 °C for 10 min (A) and 30 min (B) and then analyzed. C, quantification of the data in A and B with S.D. based on three independent experiments. D, plot of percent of product formation as a logarithm of Rad54 concentration with indicated median effective concentration (EC50).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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fig2: Rad54 concentration-dependent enhancement of Mus81·Mms4-mediated 3′ DNA flap cleavage. Reaction mixtures containing 3′ DNA flap (6 nm), Mus81·Mms4 (0.25 nm), and the indicated amount of Rad54 (0.5, 1, 2, 4, 8, or 16 nm) were incubated at 37 °C for 10 min (A) and 30 min (B) and then analyzed. C, quantification of the data in A and B with S.D. based on three independent experiments. D, plot of percent of product formation as a logarithm of Rad54 concentration with indicated median effective concentration (EC50).
Mentions: Rad54 Stimulates Cleavage of 3′ DNA Flap—The physical interaction between Rad54 and Mus81 together with the demonstrated binding preference of Rad54 for substrates that Mus81·Mms4 acts on prompted us to test whether Rad54 might modulate the nuclease activity of the Mus81·Mms4 complex. We first used the 3′ DNA flap substrate, as it is bound by Rad54 with higher affinity and represents a relevant DNA intermediate that arises during DNA repair or replication. We performed a Rad54 protein titration with 0.25 nm Mus81·Mms4, an amount that could cleave only a small fraction of the substrate (supplemental Fig. 2, A and C). Importantly, strong enhancement of the Mus81·Mms4 DNA cleavage activity occurred in a Rad54 protein concentration-dependent manner. Flap cleavage was stimulated 3-fold by 1 nm Rad54 and 5-fold by 2 nm Rad54, and complete incision of the substrate was seen when 8 nm Rad54 was added (Fig. 2, A–C). The plot of the percent product formed as a logarithmic function of Rad54 concentration yields a sigmoid curve for stimulation, with the median effective concentration EC50 = 1.56 nm (Fig. 2D). This Rad54 amount corresponds to 6 times that of Mus81·Mms4 heterodimer. Because Rad54 forms oligomeric complexes on DNA (22, 29, 30), our results are consistent with the premise that optimal stimulation of Mus81·Mms4 occurs upon assembly of a Rad54 oligomer. As expected, Rad54 alone was devoid of nucleolytic activity (Fig. 2, A and B, lane 9). Time course experiments provided further details on the effect of Rad54 on the rate of Mus81·Mms4-mediated DNA cleavage (Fig. 3). We note that the presence of Rad54 does not affect the position of the cleavage site within the flap structure (data not shown). In addition, the presence of His6 affinity tag on Mms4 does not have any significant effect on the nuclease activity of Mus81·Mms4 or on the enhancement of substrate cleavage by Rad54 (supplemental Fig. 3). Taken together, the results demonstrate that Rad54 can strongly stimulate the ability of Mus81·Mms4 to cleave the 3′ DNA flap structure and also suggest that optimal enhancement is contingent upon oligomerization of Rad54.

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