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. 2020 Jul 1;48(14):7818–7833. doi: 10.1093/nar/gkaa555

Figure 5.

Figure 5.

DSS1 and ssDNA disrupt BRCA2 self-association. (A) DSS1 inhibits the interactions between the C-terminus of BRCA2 (MBP-BRCA2CT) and full-length BRCA2 (GST-BRCA2). The reactions contained 1.5 nM GST-BRCA2, 15 nM MBP-BRCA2CT, and 1.5 μM DSS1. (B) A stoichiometric amount of DSS1 inhibits the self-interaction between the N-terminus of BRCA2 (GST-BRCA2NT) and C-terminus of BRCA2 (MBP-BRCA2CT). The reactions contained 30 nM GST-BRCA2NT and 100 nM MBP-BRCA2CT, with 0, 0.1, 0.5, 1 and 5 μM DSS1. (C) Sequence alignment of DSS1 proteins from different species. Conserved acidic regions 1 and 2 and the poorly conserved linker are labeled. Asterisks indicated the conserved residues in all shown species. The sequences of DSS1 halves (DSS1-N and DSS1-C) are illustrated. (D) DSS1 halves partially inhibit the self-interaction between the N-terminus of BRCA2 (GST-BRCA2NT) and C-terminus of BRCA2 (MBP-BRCA2CT). The reactions contained 30 nM GST-BRCA2NT, 100 nM MBP-BRCA2CT, and 0.5 μM DSS1, or 1 μM DSS1-N, or 1 μM DSS1-C. (E) Both DSS1 and ssDNA inhibits the N-to-C terminal interaction, and the inhibition from ssDNA has a requirement in length. The reactions contained 30 nM GST-BRCA2NT, 100 nM MBP-BRCA2CT, and/or 0.5 μM DSS1, 0.5 μM 100mer (olWDH1734), or 0.5 μM 20mer (olWDH1728). Quantitation is shown below. Values are expressed as mean and SD (n = 3). P values were determined with a two-tailed t-test. *P<0.05, n.s: no significance. (F) The inhibition of ssDNA on the self-interaction between the N- and C-terminus of BRCA2 increases with its length. (G) The inhibition of ssDNA on the N-to-N interaction of BRCA2 also depends on its length. The reactions contained 30 nM GST-BRCA2NT, 100 nM MBP-BRCA2CT in F or 100 nM MBP-BRCA2NT in G, and 0.5 μM ssDNA with indicated length.