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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

Interactions of Rad55–Rad57 with the Shu complex and Rad52.(a) Rad55–Rad57 was incubated with Csm2–Psy3 and the protein complex was captured through the (His)6 tag on Rad55 using Ni2+ resin. E, eluate from the resin; S, supernatant containing unbound proteins; W, wash of the resin. (b) Rad52 was tested for Y2H interaction with the indicated proteins. (c) The Y2H interaction between Rad52 and Csm2 was examined in wild-type or rad55Δ cells. (d) The Y2H interaction between Rad52 and Rad55 was examined in wild-type or csm2Δ cells. (e) GST-tagged Rad52 was mixed with Rad55–Rad57, Shu complex, or both, and protein complexes were captured on glutathione resin. Immunoblotting for the FLAG tag on Rad57 and Psy3 was used to identify Rad55–Rad57 and Shu complex retained on the resin.
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f1: Interactions of Rad55–Rad57 with the Shu complex and Rad52.(a) Rad55–Rad57 was incubated with Csm2–Psy3 and the protein complex was captured through the (His)6 tag on Rad55 using Ni2+ resin. E, eluate from the resin; S, supernatant containing unbound proteins; W, wash of the resin. (b) Rad52 was tested for Y2H interaction with the indicated proteins. (c) The Y2H interaction between Rad52 and Csm2 was examined in wild-type or rad55Δ cells. (d) The Y2H interaction between Rad52 and Rad55 was examined in wild-type or csm2Δ cells. (e) GST-tagged Rad52 was mixed with Rad55–Rad57, Shu complex, or both, and protein complexes were captured on glutathione resin. Immunoblotting for the FLAG tag on Rad57 and Psy3 was used to identify Rad55–Rad57 and Shu complex retained on the resin.

Mentions: We first focused on the purified Csm2–Psy3 heterodimer (Supplementary Fig. 1a), as the known protein interaction and DNA-binding activities occur through these subunits, including our previous findings that Csm2 associates with Rad55 in the Y2H system16. Indeed, we found by affinity pull-down that Csm2–Psy3 interacts with Rad55–Rad57 (Fig. 1a and Supplementary Fig. 2a). Similarly, the Shu complex (Csm2–Psy3–Shu1–Shu2) interacts with Rad55–Rad57 by pull-down (Supplementary Fig. 2a). We next asked whether Csm2 would interact with Rad52 by Y2H analysis (Fig. 1b). As shown before, association of Csm2 with Rad55 and self-interaction of Rad52 were seen161718, and, importantly, Csm2 and Rad55 interacted with Rad52 (Fig. 1b). While the Csm2–Rad52 interaction was abolished in rad55Δ cells, the Rad52–Rad55 interaction remained without CSM2 (Fig. 1c,d). We employed biochemical pull-down to validate these interactions and found that, consistent with the Y2H results above, while GST-tagged Rad52 could interact with Rad55–Rad57 (Fig. 1e), its interaction with the Shu complex was negligible. We attempted to validate that Rad55–Rad57 bridges Rad52 to Shu complex by performing the GST pull-down on a mixture of GST–Rad52, Rad55–Rad57 and Shu complex. However, the degree to which Shu complex is pulled down by Rad52 is either not affected or only modestly affected by the presence of Rad55–Rad57 (Fig. 1e), perhaps because the associations are too labile for the tripartite assembly to persist through washing steps. In agreement with prior Y2H results1617, pull-down analysis revealed that Rad55–Rad57 interacts with Rad51 while Csm2–Psy3 does so only weakly (Supplementary Fig. 2b). Addition of Csm2–Psy3 does not attenuate Rad55–Rad57 interaction with Rad51, suggesting the interactions are non-competitive. In addition, we have tested RPA in pull-downs and found that it may interact weakly with Rad55–Rad57, though this association could not be validated by Y2H (Supplementary Fig. 2c).


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)

Interactions of Rad55–Rad57 with the Shu complex and Rad52.(a) Rad55–Rad57 was incubated with Csm2–Psy3 and the protein complex was captured through the (His)6 tag on Rad55 using Ni2+ resin. E, eluate from the resin; S, supernatant containing unbound proteins; W, wash of the resin. (b) Rad52 was tested for Y2H interaction with the indicated proteins. (c) The Y2H interaction between Rad52 and Csm2 was examined in wild-type or rad55Δ cells. (d) The Y2H interaction between Rad52 and Rad55 was examined in wild-type or csm2Δ cells. (e) GST-tagged Rad52 was mixed with Rad55–Rad57, Shu complex, or both, and protein complexes were captured on glutathione resin. Immunoblotting for the FLAG tag on Rad57 and Psy3 was used to identify Rad55–Rad57 and Shu complex retained on the resin.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4525180&req=5

f1: Interactions of Rad55–Rad57 with the Shu complex and Rad52.(a) Rad55–Rad57 was incubated with Csm2–Psy3 and the protein complex was captured through the (His)6 tag on Rad55 using Ni2+ resin. E, eluate from the resin; S, supernatant containing unbound proteins; W, wash of the resin. (b) Rad52 was tested for Y2H interaction with the indicated proteins. (c) The Y2H interaction between Rad52 and Csm2 was examined in wild-type or rad55Δ cells. (d) The Y2H interaction between Rad52 and Rad55 was examined in wild-type or csm2Δ cells. (e) GST-tagged Rad52 was mixed with Rad55–Rad57, Shu complex, or both, and protein complexes were captured on glutathione resin. Immunoblotting for the FLAG tag on Rad57 and Psy3 was used to identify Rad55–Rad57 and Shu complex retained on the resin.
Mentions: We first focused on the purified Csm2–Psy3 heterodimer (Supplementary Fig. 1a), as the known protein interaction and DNA-binding activities occur through these subunits, including our previous findings that Csm2 associates with Rad55 in the Y2H system16. Indeed, we found by affinity pull-down that Csm2–Psy3 interacts with Rad55–Rad57 (Fig. 1a and Supplementary Fig. 2a). Similarly, the Shu complex (Csm2–Psy3–Shu1–Shu2) interacts with Rad55–Rad57 by pull-down (Supplementary Fig. 2a). We next asked whether Csm2 would interact with Rad52 by Y2H analysis (Fig. 1b). As shown before, association of Csm2 with Rad55 and self-interaction of Rad52 were seen161718, and, importantly, Csm2 and Rad55 interacted with Rad52 (Fig. 1b). While the Csm2–Rad52 interaction was abolished in rad55Δ cells, the Rad52–Rad55 interaction remained without CSM2 (Fig. 1c,d). We employed biochemical pull-down to validate these interactions and found that, consistent with the Y2H results above, while GST-tagged Rad52 could interact with Rad55–Rad57 (Fig. 1e), its interaction with the Shu complex was negligible. We attempted to validate that Rad55–Rad57 bridges Rad52 to Shu complex by performing the GST pull-down on a mixture of GST–Rad52, Rad55–Rad57 and Shu complex. However, the degree to which Shu complex is pulled down by Rad52 is either not affected or only modestly affected by the presence of Rad55–Rad57 (Fig. 1e), perhaps because the associations are too labile for the tripartite assembly to persist through washing steps. In agreement with prior Y2H results1617, pull-down analysis revealed that Rad55–Rad57 interacts with Rad51 while Csm2–Psy3 does so only weakly (Supplementary Fig. 2b). Addition of Csm2–Psy3 does not attenuate Rad55–Rad57 interaction with Rad51, suggesting the interactions are non-competitive. In addition, we have tested RPA in pull-downs and found that it may interact weakly with Rad55–Rad57, though this association could not be validated by Y2H (Supplementary Fig. 2c).

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