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Promotion of presynaptic filament assembly by the ensemble of S. cerevisiae Rad51 paralogues with Rad52.

Gaines WA, Godin SK, Kabbinavar FF, Rao T, VanDemark AP, Sung P, Bernstein KA - Nat Commun (2015)

Bottom Line: Rad55 and Rad57 are associated in a heterodimer, while Csm2 and Psy3 form the Shu complex with Shu1 and Shu2.The csm2-F46A allele is unable to interact with Rad55, ablating the ability of the Shu complex to enhance Rad51 presynaptic filament assembly in vitro and impairing HR in vivo.Our results reveal that Rad55-Rad57, the Shu complex and Rad52 act as a functional ensemble to promote Rad51-filament assembly, which has important implications for understanding the role of the human RAD51 paralogues in Fanconi anaemia and cancer predisposition.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biochemistry and Biophysics, Yale University School of Medicine, New Haven, Conneticut 06510, USA.

ABSTRACT
The conserved budding yeast Rad51 paralogues, including Rad55, Rad57, Csm2 and Psy3 are indispensable for homologous recombination (HR)-mediated chromosome damage repair. Rad55 and Rad57 are associated in a heterodimer, while Csm2 and Psy3 form the Shu complex with Shu1 and Shu2. Here we show that Rad55 bridges an interaction between Csm2 with Rad51 and Rad52 and, using a fully reconstituted system, demonstrate that the Shu complex synergizes with Rad55-Rad57 and Rad52 to promote nucleation of Rad51 on single-stranded DNA pre-occupied by replication protein A (RPA). The csm2-F46A allele is unable to interact with Rad55, ablating the ability of the Shu complex to enhance Rad51 presynaptic filament assembly in vitro and impairing HR in vivo. Our results reveal that Rad55-Rad57, the Shu complex and Rad52 act as a functional ensemble to promote Rad51-filament assembly, which has important implications for understanding the role of the human RAD51 paralogues in Fanconi anaemia and cancer predisposition.

No MeSH data available.


Related in: MedlinePlus

Impairment of homologous recombination by the csm2–F46A mutation.(a) csm2–F46A cells are as sensitive to MMS as a csm2Δ mutant. Cultures were fivefold serially diluted onto YPD medium with the indicated dose of MMS and incubated for 2 days at 30 °C. (b) Similar to csm2Δ, csm2–F46A alleviates the MMS and HU sensitivity of an sgs1Δ mutant. Cells of the indicated genotypes were fivefold serially diluted and tested for sensitivity to 0.012% MMS or 100 mM HU. (c) WT, csm2Δ or csm2-F64A cells harbouring a direct repeat HR reporter (leu2-ΔEcoRI::URA3::leu2-ΔBstEII) were tested for spontaneous rates of Rad51-dependent gene conversion (GC) and Rad51-independent single-strand annealing (SSA) as described16. The rates of GC are significantly decreased in both csm2–F46A and csm2Δ strains (P<0.01 by student's t-test) with a corresponding increase in SSA relative to WT strains (P<0.02 by student's t-test). S.d. are plotted as the error bars (n=3) and ‘*' indicates significance. (d) Like csm2Δ cells, csm2–F46A cells exhibit an elevated mutation rate in a canavanine mutagenesis assay. S.d. are plotted as error bars (n=5) and ‘*' indicates significance.
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f4: Impairment of homologous recombination by the csm2–F46A mutation.(a) csm2–F46A cells are as sensitive to MMS as a csm2Δ mutant. Cultures were fivefold serially diluted onto YPD medium with the indicated dose of MMS and incubated for 2 days at 30 °C. (b) Similar to csm2Δ, csm2–F46A alleviates the MMS and HU sensitivity of an sgs1Δ mutant. Cells of the indicated genotypes were fivefold serially diluted and tested for sensitivity to 0.012% MMS or 100 mM HU. (c) WT, csm2Δ or csm2-F64A cells harbouring a direct repeat HR reporter (leu2-ΔEcoRI::URA3::leu2-ΔBstEII) were tested for spontaneous rates of Rad51-dependent gene conversion (GC) and Rad51-independent single-strand annealing (SSA) as described16. The rates of GC are significantly decreased in both csm2–F46A and csm2Δ strains (P<0.01 by student's t-test) with a corresponding increase in SSA relative to WT strains (P<0.02 by student's t-test). S.d. are plotted as the error bars (n=3) and ‘*' indicates significance. (d) Like csm2Δ cells, csm2–F46A cells exhibit an elevated mutation rate in a canavanine mutagenesis assay. S.d. are plotted as error bars (n=5) and ‘*' indicates significance.

Mentions: To determine if the Csm2–Rad55 interaction is important for HR, we integrated csm2–F46A into the endogenous CSM2 locus and found that the mutant cells are almost as sensitive to MMS as csm2Δ cells (Fig. 4a). Furthermore, since previous studies demonstrated that the Shu complex acts upstream of Sgs1, we tested if the csm2–F46A mutation suppresses the MMS and HU sensitivity of an sgs1Δ mutant like csm2Δ and found that it does (Fig. 4b). We next employed a direct repeat recombination assay1631 to reveal that csm2–F46A cells are as impaired as the csm2Δ mutant in Rad51-mediated gene conversion (Fig. 4c), with a corresponding increase in Rad51-independent single-strand annealing events (Fig. 4c16; P value<0.02 by student's t-test). Consistent with disruption of other HR factors32, using a canavanine mutagenesis assay we found that csm2–F46A cells accumulate spontaneous mutations like csm2Δ (refs 10, 13) (Fig. 4d). Together, our results demonstrate that the interaction between Csm2 and Rad55 is indispensable for the biological efficacy of the Shu complex.


Promotion of presynaptic filament assembly by the ensemble of S. cerevisiae Rad51 paralogues with Rad52.

Gaines WA, Godin SK, Kabbinavar FF, Rao T, VanDemark AP, Sung P, Bernstein KA - Nat Commun (2015)

Impairment of homologous recombination by the csm2–F46A mutation.(a) csm2–F46A cells are as sensitive to MMS as a csm2Δ mutant. Cultures were fivefold serially diluted onto YPD medium with the indicated dose of MMS and incubated for 2 days at 30 °C. (b) Similar to csm2Δ, csm2–F46A alleviates the MMS and HU sensitivity of an sgs1Δ mutant. Cells of the indicated genotypes were fivefold serially diluted and tested for sensitivity to 0.012% MMS or 100 mM HU. (c) WT, csm2Δ or csm2-F64A cells harbouring a direct repeat HR reporter (leu2-ΔEcoRI::URA3::leu2-ΔBstEII) were tested for spontaneous rates of Rad51-dependent gene conversion (GC) and Rad51-independent single-strand annealing (SSA) as described16. The rates of GC are significantly decreased in both csm2–F46A and csm2Δ strains (P<0.01 by student's t-test) with a corresponding increase in SSA relative to WT strains (P<0.02 by student's t-test). S.d. are plotted as the error bars (n=3) and ‘*' indicates significance. (d) Like csm2Δ cells, csm2–F46A cells exhibit an elevated mutation rate in a canavanine mutagenesis assay. S.d. are plotted as error bars (n=5) and ‘*' indicates significance.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4525180&req=5

f4: Impairment of homologous recombination by the csm2–F46A mutation.(a) csm2–F46A cells are as sensitive to MMS as a csm2Δ mutant. Cultures were fivefold serially diluted onto YPD medium with the indicated dose of MMS and incubated for 2 days at 30 °C. (b) Similar to csm2Δ, csm2–F46A alleviates the MMS and HU sensitivity of an sgs1Δ mutant. Cells of the indicated genotypes were fivefold serially diluted and tested for sensitivity to 0.012% MMS or 100 mM HU. (c) WT, csm2Δ or csm2-F64A cells harbouring a direct repeat HR reporter (leu2-ΔEcoRI::URA3::leu2-ΔBstEII) were tested for spontaneous rates of Rad51-dependent gene conversion (GC) and Rad51-independent single-strand annealing (SSA) as described16. The rates of GC are significantly decreased in both csm2–F46A and csm2Δ strains (P<0.01 by student's t-test) with a corresponding increase in SSA relative to WT strains (P<0.02 by student's t-test). S.d. are plotted as the error bars (n=3) and ‘*' indicates significance. (d) Like csm2Δ cells, csm2–F46A cells exhibit an elevated mutation rate in a canavanine mutagenesis assay. S.d. are plotted as error bars (n=5) and ‘*' indicates significance.
Mentions: To determine if the Csm2–Rad55 interaction is important for HR, we integrated csm2–F46A into the endogenous CSM2 locus and found that the mutant cells are almost as sensitive to MMS as csm2Δ cells (Fig. 4a). Furthermore, since previous studies demonstrated that the Shu complex acts upstream of Sgs1, we tested if the csm2–F46A mutation suppresses the MMS and HU sensitivity of an sgs1Δ mutant like csm2Δ and found that it does (Fig. 4b). We next employed a direct repeat recombination assay1631 to reveal that csm2–F46A cells are as impaired as the csm2Δ mutant in Rad51-mediated gene conversion (Fig. 4c), with a corresponding increase in Rad51-independent single-strand annealing events (Fig. 4c16; P value<0.02 by student's t-test). Consistent with disruption of other HR factors32, using a canavanine mutagenesis assay we found that csm2–F46A cells accumulate spontaneous mutations like csm2Δ (refs 10, 13) (Fig. 4d). Together, our results demonstrate that the interaction between Csm2 and Rad55 is indispensable for the biological efficacy of the Shu complex.

Bottom Line: Rad55 and Rad57 are associated in a heterodimer, while Csm2 and Psy3 form the Shu complex with Shu1 and Shu2.The csm2-F46A allele is unable to interact with Rad55, ablating the ability of the Shu complex to enhance Rad51 presynaptic filament assembly in vitro and impairing HR in vivo.Our results reveal that Rad55-Rad57, the Shu complex and Rad52 act as a functional ensemble to promote Rad51-filament assembly, which has important implications for understanding the role of the human RAD51 paralogues in Fanconi anaemia and cancer predisposition.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biochemistry and Biophysics, Yale University School of Medicine, New Haven, Conneticut 06510, USA.

ABSTRACT
The conserved budding yeast Rad51 paralogues, including Rad55, Rad57, Csm2 and Psy3 are indispensable for homologous recombination (HR)-mediated chromosome damage repair. Rad55 and Rad57 are associated in a heterodimer, while Csm2 and Psy3 form the Shu complex with Shu1 and Shu2. Here we show that Rad55 bridges an interaction between Csm2 with Rad51 and Rad52 and, using a fully reconstituted system, demonstrate that the Shu complex synergizes with Rad55-Rad57 and Rad52 to promote nucleation of Rad51 on single-stranded DNA pre-occupied by replication protein A (RPA). The csm2-F46A allele is unable to interact with Rad55, ablating the ability of the Shu complex to enhance Rad51 presynaptic filament assembly in vitro and impairing HR in vivo. Our results reveal that Rad55-Rad57, the Shu complex and Rad52 act as a functional ensemble to promote Rad51-filament assembly, which has important implications for understanding the role of the human RAD51 paralogues in Fanconi anaemia and cancer predisposition.

No MeSH data available.


Related in: MedlinePlus