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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 physical interaction and functional synergy of Shu complex with Rad55–Rad57 by the csm2–F46A mutation.(a) Cartoon view of the Csm2–Psy3 heterodimer29, with Csm2 in blue and Psy3 in green. Csm2–F46 is highlighted in red. (b) Y2H analysis for interaction of Csm2 and csm2–F46A with Rad55, Rad51, Rad52 and Psy3. (c) Pull-down assay (as in Fig. 1a) to examine interaction of (His)6-Rad55–Rad57 with Csm2–Psy3 harbouring csm2–F46A. (d) Analysis of the DNA-binding activity of Shu complex harbouring csm2–F46A. (e) Rad51 loading and (f) DNA strand exchange assays were carried out as in Fig. 2 to evaluate the efficacy of Shu complex harbouring csm2–F46A in the promotion of presynaptic filament assembly. S.d. are plotted as error bars (n=3) and ‘*' indicates significance.
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f3: Impairment of physical interaction and functional synergy of Shu complex with Rad55–Rad57 by the csm2–F46A mutation.(a) Cartoon view of the Csm2–Psy3 heterodimer29, with Csm2 in blue and Psy3 in green. Csm2–F46 is highlighted in red. (b) Y2H analysis for interaction of Csm2 and csm2–F46A with Rad55, Rad51, Rad52 and Psy3. (c) Pull-down assay (as in Fig. 1a) to examine interaction of (His)6-Rad55–Rad57 with Csm2–Psy3 harbouring csm2–F46A. (d) Analysis of the DNA-binding activity of Shu complex harbouring csm2–F46A. (e) Rad51 loading and (f) DNA strand exchange assays were carried out as in Fig. 2 to evaluate the efficacy of Shu complex harbouring csm2–F46A in the promotion of presynaptic filament assembly. S.d. are plotted as error bars (n=3) and ‘*' indicates significance.

Mentions: By X-ray crystallography, several groups have determined the structure of Csm2–Psy3 (refs 27, 28, 29). We searched for solvent exposed residues that could participate in interaction with a partner molecule such as Rad55. We applied surface triplet propensity analysis30 to this structure29 to reveal a potential protein interaction surface on Csm2 (Supplementary Fig. 7a), which is highly conserved in numerous fungal homologues (Supplementary Fig. 7b). The phenylalanine 46 to alanine (F46A) (Fig. 3a and Supplementary Fig. 7) mutation was made, as changing this surface residue is unlikely to affect the stability or folding of Csm2. Importantly, we found that the F46A mutation disrupts the Y2H interaction of Csm2 with Rad55 but not Psy3 (Fig. 3b).


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 physical interaction and functional synergy of Shu complex with Rad55–Rad57 by the csm2–F46A mutation.(a) Cartoon view of the Csm2–Psy3 heterodimer29, with Csm2 in blue and Psy3 in green. Csm2–F46 is highlighted in red. (b) Y2H analysis for interaction of Csm2 and csm2–F46A with Rad55, Rad51, Rad52 and Psy3. (c) Pull-down assay (as in Fig. 1a) to examine interaction of (His)6-Rad55–Rad57 with Csm2–Psy3 harbouring csm2–F46A. (d) Analysis of the DNA-binding activity of Shu complex harbouring csm2–F46A. (e) Rad51 loading and (f) DNA strand exchange assays were carried out as in Fig. 2 to evaluate the efficacy of Shu complex harbouring csm2–F46A in the promotion of presynaptic filament assembly. S.d. are plotted as error bars (n=3) and ‘*' indicates significance.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Impairment of physical interaction and functional synergy of Shu complex with Rad55–Rad57 by the csm2–F46A mutation.(a) Cartoon view of the Csm2–Psy3 heterodimer29, with Csm2 in blue and Psy3 in green. Csm2–F46 is highlighted in red. (b) Y2H analysis for interaction of Csm2 and csm2–F46A with Rad55, Rad51, Rad52 and Psy3. (c) Pull-down assay (as in Fig. 1a) to examine interaction of (His)6-Rad55–Rad57 with Csm2–Psy3 harbouring csm2–F46A. (d) Analysis of the DNA-binding activity of Shu complex harbouring csm2–F46A. (e) Rad51 loading and (f) DNA strand exchange assays were carried out as in Fig. 2 to evaluate the efficacy of Shu complex harbouring csm2–F46A in the promotion of presynaptic filament assembly. S.d. are plotted as error bars (n=3) and ‘*' indicates significance.
Mentions: By X-ray crystallography, several groups have determined the structure of Csm2–Psy3 (refs 27, 28, 29). We searched for solvent exposed residues that could participate in interaction with a partner molecule such as Rad55. We applied surface triplet propensity analysis30 to this structure29 to reveal a potential protein interaction surface on Csm2 (Supplementary Fig. 7a), which is highly conserved in numerous fungal homologues (Supplementary Fig. 7b). The phenylalanine 46 to alanine (F46A) (Fig. 3a and Supplementary Fig. 7) mutation was made, as changing this surface residue is unlikely to affect the stability or folding of Csm2. Importantly, we found that the F46A mutation disrupts the Y2H interaction of Csm2 with Rad55 but not Psy3 (Fig. 3b).

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