Figure 7
Figure 7. Effect of BRCA2 variants on the structure of the C-terminal domain of mouse BRCA2 and analysis of BRCA2-DSS1 interaction. (A-B) Human BRCA2 mutations in C-terminal region and the corresponding mutations in the mouse BRCA2 are listed in the table. Structure of C-terminal region of BRCA2 that includes 3 oligonucleotide/oligosaccharide-binding (OB) domains OB1, OB2, and OB3, preceded by a helix-rich helical domain, whereas the helical domain (A) and OB1 interact with an essential cofactor DSS1 (B). The mutations studied in this work are present in the OB1 and the helical domain, whose side chains are colored magenta (if present in the helical domain) or cyan (if present in the OB1 domain) and highlighted in green. The effects of variants on the structure of BRCA2 described here are computer predictions and not primary crystallographic data. (C-D) Leucine at position 2411 is present at the first helix of helical domain (C). Its side chain is colored magenta, and its mutant is shown in Thr2411 (D). (E) Leucine at position 2431 is present at the core of the helical domain and interacts with DSS1. The side chain of Leu2431 is displayed, and its surface is colored pink. The protein DSS1 surface is displayed and colored white, except the hydrophobic patch between Leu10 and Leu12 of DSS1 is colored green. (F) The mutant of the same residue, Pro2431, surface is displayed in white. (G) Tryptophan at position 2547 is present within the helical domain and crucially placed at the interface of this domain and OB1. Trp2547 makes several van-der Waals interactions with Thr2643 of the OB1 domain, shown at yellow dotted lines. (H) A cysteine mutant Cys2547 side chain is shown as stick diagram, which might have the tendency to form a disulfide bond with a cysteine residue Cys2610. (I) Arginine at position 2650 is present at the surface of the OB1 domain (blue sticks), which forms a hydrogen bond with the backbone of Asp2693, whereas mutant Asn2650 loses the hydrogen bond (J). (K) Coimmunoprecipitation experiments of extracts after transient expression of DSS1-GFP construct in ES cells expressing either WT or mutant BRCA2. DSS1-GFP was detected by WB in immunoprecipitations (IP) with c-myc antibody. BRCA2 is tagged with c-myc, and DSS1 is tagged with GFP. Note that, in p.L2510P mutants, DSS1-GFP IP was reduced (lane 3), but in p.W2626C (lane 5), p.K2729N (lane 7), and p.I2490T (lane 9) mutants, the amount of DSS1 immunoprecipitated with BRCA2 is comparable to the WT (lane 1). Actin was used for input control. (L) IP using GFP antibody. WB using c-myc antibody after performing IP with GFP antibody detected BRCA2. DSS1-GFP was detected by DSS1 antibody. The amount of immunoprecipitated BRCA2 is reduced in p.L2510P mutants (lane 3), but not in p.W2626C (lane 5), p.K2729N (lane 7), and p.I2490T (lane 9) compared with the WT cells (lane 1). Actin was used to show the same amount of protein used in the IP experiments.

Effect of BRCA2 variants on the structure of the C-terminal domain of mouse BRCA2 and analysis of BRCA2-DSS1 interaction. (A-B) Human BRCA2 mutations in C-terminal region and the corresponding mutations in the mouse BRCA2 are listed in the table. Structure of C-terminal region of BRCA2 that includes 3 oligonucleotide/oligosaccharide-binding (OB) domains OB1, OB2, and OB3, preceded by a helix-rich helical domain, whereas the helical domain (A) and OB1 interact with an essential cofactor DSS1 (B). The mutations studied in this work are present in the OB1 and the helical domain, whose side chains are colored magenta (if present in the helical domain) or cyan (if present in the OB1 domain) and highlighted in green. The effects of variants on the structure of BRCA2 described here are computer predictions and not primary crystallographic data. (C-D) Leucine at position 2411 is present at the first helix of helical domain (C). Its side chain is colored magenta, and its mutant is shown in Thr2411 (D). (E) Leucine at position 2431 is present at the core of the helical domain and interacts with DSS1. The side chain of Leu2431 is displayed, and its surface is colored pink. The protein DSS1 surface is displayed and colored white, except the hydrophobic patch between Leu10 and Leu12 of DSS1 is colored green. (F) The mutant of the same residue, Pro2431, surface is displayed in white. (G) Tryptophan at position 2547 is present within the helical domain and crucially placed at the interface of this domain and OB1. Trp2547 makes several van-der Waals interactions with Thr2643 of the OB1 domain, shown at yellow dotted lines. (H) A cysteine mutant Cys2547 side chain is shown as stick diagram, which might have the tendency to form a disulfide bond with a cysteine residue Cys2610. (I) Arginine at position 2650 is present at the surface of the OB1 domain (blue sticks), which forms a hydrogen bond with the backbone of Asp2693, whereas mutant Asn2650 loses the hydrogen bond (J). (K) Coimmunoprecipitation experiments of extracts after transient expression of DSS1-GFP construct in ES cells expressing either WT or mutant BRCA2. DSS1-GFP was detected by WB in immunoprecipitations (IP) with c-myc antibody. BRCA2 is tagged with c-myc, and DSS1 is tagged with GFP. Note that, in p.L2510P mutants, DSS1-GFP IP was reduced (lane 3), but in p.W2626C (lane 5), p.K2729N (lane 7), and p.I2490T (lane 9) mutants, the amount of DSS1 immunoprecipitated with BRCA2 is comparable to the WT (lane 1). Actin was used for input control. (L) IP using GFP antibody. WB using c-myc antibody after performing IP with GFP antibody detected BRCA2. DSS1-GFP was detected by DSS1 antibody. The amount of immunoprecipitated BRCA2 is reduced in p.L2510P mutants (lane 3), but not in p.W2626C (lane 5), p.K2729N (lane 7), and p.I2490T (lane 9) compared with the WT cells (lane 1). Actin was used to show the same amount of protein used in the IP experiments.

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