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. Author manuscript; available in PMC: 2010 Mar 20.

Published in final edited form as: Cell. 2009 Mar 20;136(6):1032–1043. doi: 10.1016/j.cell.2009.02.019

(A) Gel showing RAD51 (3 μM) incubated with either GST-BRC4 or GST-Δ1524-30 prior to incubation with ³²P-labeled dT₄₀ ssDNA (0.3 μM) for 1 h in the presence of ATP, Mg²⁺ and Ca²⁺ (B) Quantification of protein-DNA complexes with GST-BRC4 (filled circles) or GST-Δ1524-30 (open circles) obtained as in (A), using: ATP, Mg²⁺ + Ca²⁺ (●); ATP and Mg²⁺ (); ADP, Mg²⁺ + Ca²⁺ (), ATP and Ca²⁺ (); AMP-PNP, Mg²⁺ + Ca²⁺ (); or no nucleotide cofactor, Mg²⁺ + Ca²⁺ (). Data obtained with the control mutant, GST-T1526A, ATP, Mg²⁺ + Ca²⁺: (). (C) BRC4 forms a stoichiometric complex with RAD51-ssDNA complexes. RAD51 (0.15 μM) was incubated with BRC4 () or BRCA2_BRC1-8 (), or RAD51 (0.3 μM) was incubated with BRC4 (●), prior to incubation with ³²P-labeled dT₄₀ ssDNA (15 nM) for another 15 min in the presence of ATP, Mg²⁺ and Ca²⁺. Due to limitations of the protein stock, higher BRCA2_BRC1-8 concentrations could not be examined. (D) BRC4 increases the stability of RAD51-ssDNA complexes to increasing concentrations salt: complexes were formed with RAD51 (0.9 μM) alone (upper panel) or RAD51 (0.9 μM) and GST-BRC4 (3.6 μM) (lower panel), in the presence of ADP and Mg²⁺.

Inline graphic — (A) Gel showing RAD51 (3 μM) incubated with either GST-BRC4 or GST-Δ1524-30 prior to incubation with ³²P-labeled dT₄₀ ssDNA (0.3 μM) for 1 h in the presence of ATP, Mg²⁺ and Ca²⁺ (B) Quantification of protein-DNA complexes with GST-BRC4 (filled circles) or GST-Δ1524-30 (open circles) obtained as in (A), using: ATP, Mg²⁺ + Ca²⁺ (●); ATP and Mg²⁺ (); ADP, Mg²⁺ + Ca²⁺ (), ATP and Ca²⁺ (); AMP-PNP, Mg²⁺ + Ca²⁺ (); or no nucleotide cofactor, Mg²⁺ + Ca²⁺ (). Data obtained with the control mutant, GST-T1526A, ATP, Mg²⁺ + Ca²⁺: (). (C) BRC4 forms a stoichiometric complex with RAD51-ssDNA complexes. RAD51 (0.15 μM) was incubated with BRC4 () or BRCA2_BRC1-8 (), or RAD51 (0.3 μM) was incubated with BRC4 (●), prior to incubation with ³²P-labeled dT₄₀ ssDNA (15 nM) for another 15 min in the presence of ATP, Mg²⁺ and Ca²⁺. Due to limitations of the protein stock, higher BRCA2_BRC1-8 concentrations could not be examined. (D) BRC4 increases the stability of RAD51-ssDNA complexes to increasing concentrations salt: complexes were formed with RAD51 (0.9 μM) alone (upper panel) or RAD51 (0.9 μM) and GST-BRC4 (3.6 μM) (lower panel), in the presence of ADP and Mg²⁺.