• Home
  • Search Results
  • Promotion of presynaptic filament assembly by the ensemble of S. cerevisiae Rad51 paralogues with Rad52.

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

Nature communications (2015-07-29)
William A Gaines, Stephen K Godin, Faiz F Kabbinavar, Timsi Rao, Andrew P VanDemark, Patrick Sung, Kara A Bernstein
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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Glycerol, for molecular biology, ≥99.0%
Sigma-Aldrich
Magnesium chloride, anhydrous, ≥98%
Sigma-Aldrich
Glycerol, ≥99.5%
Sigma-Aldrich
Magnesium chloride solution, for molecular biology, 1.00 M±0.01 M
Sigma-Aldrich
MOPS, ≥99.5% (titration)
Sigma-Aldrich
Imidazole, ACS reagent, ≥99% (titration)
Sigma-Aldrich
Ethylenediaminetetraacetic acid, ACS reagent, 99.4-100.6%, powder
Sigma-Aldrich
Phenylmethanesulfonyl fluoride, ≥98.5% (GC)
Sigma-Aldrich
DL-Dithiothreitol solution, BioUltra, for molecular biology, ~1 M in H2O
Sigma-Aldrich
Ethylenediaminetetraacetic acid, anhydrous, BioUltra, ≥99% (titration)
Supelco
DL-Dithiothreitol solution, 1 M in H2O
Sigma-Aldrich
Imidazole, ReagentPlus®, 99%
Sigma-Aldrich
Glycerol, BioReagent, suitable for cell culture, suitable for insect cell culture, suitable for electrophoresis, ≥99% (GC)
Sigma-Aldrich
Imidazole, for molecular biology, ≥99% (titration)
Sigma-Aldrich
Monoclonal ANTI-FLAG® M2-Peroxidase (HRP) antibody produced in mouse, clone M2, purified immunoglobulin, buffered aqueous glycerol solution
Sigma-Aldrich
Imidazole, puriss. p.a., ≥99.5% (GC)
Sigma-Aldrich
Ethylenediaminetetraacetic acid, anhydrous, crystalline, BioReagent, suitable for cell culture
Sigma-Aldrich
Ethylenediaminetetraacetic acid solution, 0.02% in DPBS (0.5 mM), sterile-filtered, BioReagent, suitable for cell culture
Sigma-Aldrich
MOPS, BioPerformance Certified, suitable for cell culture, ≥99.5% (titration)
Sigma-Aldrich
Ethylenediaminetetraacetic acid, purified grade, ≥98.5%, powder
Sigma-Aldrich
Magnesium chloride, powder, <200 μm
Sigma-Aldrich
Glycerin, meets USP testing specifications
Sigma-Aldrich
Glycerol, BioXtra, ≥99% (GC)
Sigma-Aldrich
Magnesium chloride solution, BioUltra, for molecular biology, ~1 M in H2O
Sigma-Aldrich
Glycerol solution, 83.5-89.5% (T)
Sigma-Aldrich
Imidazole, BioUltra, ≥99.5% (GC)
Sigma-Aldrich
Imidazole buffer Solution, BioUltra, 1 M in H2O
Sigma-Aldrich
Phenylmethanesulfonyl fluoride, ≥99.0% (T)
Sigma-Aldrich
Ethylenediaminetetraacetic acid, 99.995% trace metals basis
Sigma-Aldrich
Glycerol, FCC, FG