Volume 7, Issue 2, Pages 273-282 (February 2001) Role of BRCA2 in Control of the RAD51 Recombination and DNA Repair Protein  Adelina A Davies, Jean-Yves Masson, Michael J McIlwraith, Alicja Z Stasiak, Andrzej Stasiak, Ashok R Venkitaraman, Stephen C West  Molecular Cell  Volume 7, Issue 2, Pages 273-282 (February 2001) DOI: 10.1016/S1097-2765(01)00175-7

Figure 1 Inhibition of RAD51–DNA Complex Formation by Peptides Corresponding to the BRC3 Region of BRCA2 (A) Schematic diagram of the BRCA2 protein, highlighting the BRC repeats that interact with RAD51. The amino acid sequence of the 69 amino acid peptide corresponding to the BRC3 repeat (amino acids 1415–1483) is indicated. The most conserved part of the BRC3 motif is shaded in gray. Three mutant peptides are also shown, and altered or deleted amino acids are indicated in red. (B) Purified RAD51 protein was incubated alone (lanes b–e) or with BRC3 (lanes g–j) prior to the addition of 32P-labeled tailed linear duplex DNA. Protein–DNA complexes were fixed and analyzed by agarose gel electrophoresis followed by autoradiography. (C) RAD51 was incubated in the absence (lane b) or presence (lanes c–i) of varying amounts of BRC3 prior to the addition of DNA Molecular Cell 2001 7, 273-282DOI: (10.1016/S1097-2765(01)00175-7)

Figure 2 Specificity of the BRC3–RAD51 Interaction (A) Human RAD51 (Hs RAD51) or Archaeoglobus fulgidus Rad51 (Af RAD51) was incubated with or without BRC3 peptide (24 μM) as indicated, and DNA binding assays were conducted as described. (B) Interactions between mutant BRC3 peptides and RAD51. RAD51 was preincubated with varying amounts of BRC3 peptide (lanes c–e), BRC3 carrying single mutations D1420Y (lanes f–h) or T1430A (lanes i–k), or BRC3 with a seven amino acid deletion ΔF1428-K1434 (lanes l–n). The products of these interactions were analyzed for their ability to bind DNA as determined by agarose gel electrophoresis Molecular Cell 2001 7, 273-282DOI: (10.1016/S1097-2765(01)00175-7)

Figure 3 Electron Microscopic Visualization of Nucleoprotein Filaments Formed by RAD51 in the Presence or Absence of BRC Peptides White arrows indicate naked duplex DNA, whereas RAD51–DNA complexes can be seen as thickened, striated nucleoprotein filaments. The black arrow indicates a ring of RAD51. All images were produced at the same magnification Molecular Cell 2001 7, 273-282DOI: (10.1016/S1097-2765(01)00175-7)

Figure 4 Effect of BRC Peptides on RAD51–DNA Complex Formation (A) The amino acid sequences of peptides (69 amino acids long) corresponding to the BRC3 repeat (amino acids 1415–1483), the BRC4 repeat (amino acids 1511–1579), and the BRC7 repeat (amino acids 1965–2033) are indicated. Sequences that are common to all three peptides are shown in red, those shared uniquely between BRC4 and BRC3 are in green, and identities between BRC4 and BRC7 are presented in blue. (B) Purified RAD51 protein (4 μM) was incubated alone (lane b) or with BRC3 peptide (lane c), BRC4 peptide (lane d), or BRC7 peptide (lane e) prior to the addition of 32P-labeled tailed linear duplex DNA. All peptides were present at 24 μM. Protein–DNA complexes were fixed and analyzed by agarose gel electrophoresis followed by autoradiography. DNA without protein (lane a) Molecular Cell 2001 7, 273-282DOI: (10.1016/S1097-2765(01)00175-7)

Figure 5 Analysis of Molecular Mass of the RAD51–BRC4 Peptide Complex RAD51 was incubated alone (lane 1) or with biotinylated BRC4 peptide (lanes 2 and 4) prior to loading onto a Superdex 200 gel filtration column. Gel filtration of the biotinylated BRC4 peptide alone is shown in lane 3. Proteins were visualized by dot blot analysis using either an anti-RAD51 antibody (lanes 1 and 2) or streptavidin-HRP (lanes 3 and 4), followed by ECL Molecular Cell 2001 7, 273-282DOI: (10.1016/S1097-2765(01)00175-7)

Figure 6 Subcellular Localization of RAD51 in BRCA2-Defective Cells Cell-free extracts were prepared from normal (MiaPaca) and BRCA2-defective (CAPAN-1) cell lines and fractionated into cytosolic (Cyt) and nuclear (Nuc) fractions. These fractions were analyzed for the presence of BRCA2 and RAD51 by SDS-PAGE followed by Western blotting Molecular Cell 2001 7, 273-282DOI: (10.1016/S1097-2765(01)00175-7)

Figure 7 Schematic Model for the Role of BRCA2 and RAD51 in DNA Repair (A) In a normal cell, RAD51 (green) and BRCA2 (pink) interact to form a complex with each other and with other proteins (blue). The complex may include proteins such as RAD52, RAD54, XRCC3, and RP-A. Upon DNA damage or replication fork breakdown, the complex is activated, possibly by posttranslational modification of BRCA2 or RAD51, and is recruited to the sites of DNA repair. There, RAD51 protein forms nucleoprotein filaments that, in conjunction with other repair proteins, effect double-strand break repair using the sister chromatid as a template. (B) In BRCA2 mutant cells typified by the BRCA2 truncation cell line CAPAN-1, complex formation between RAD51 and BRCA2 is disrupted, and much of the RAD51 resides in the cytoplasm along with the truncated BRCA2. The RAD51 that remains in the nucleus lacks BRCA2 control and may bind nonproductively at undamaged regions of DNA. In these cells, the introduction of double-strand breaks fails to stimulate the recruitment of BRCA2, RAD51, and other repair proteins, leading to inefficient homologous recombination and genomic instability Molecular Cell 2001 7, 273-282DOI: (10.1016/S1097-2765(01)00175-7)