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. 2016 Jan 1;12(2):133–143. doi: 10.7150/ijbs.14242

Figure 1.

Figure 1

BRCA1 directly binds to DNA in vitro. (A) Binding of full-length MBP-BRCA1 to dsDNA by EMSA. The identity of the samples is shown on the figure. The concentration of labeled dsDNA and protein are 40nM and 100nM, respectively. (B) Identification of the DBR of BRCA1 using GST-BRCA1 fragments. The identities of the samples are shown on the figure. The concentration of labeled dsDNA and protein are 40nM and 100nM, respectively. (C) Identification of the DBR of BRCA1 using MBP-BRCA1 fragments. The identities of the samples are shown on the figure. The concentration of labeled dsDNA and protein are 40nM and 100nM, respectively. (D) Validation of the DBR of BRCA1 using full-length BRCA1 deficient in the DBR (421-701aa) by EMSA. The identities of the samples are shown on the figure. The concentration of labeled dsDNA is 40nM. (E) In vitro stability of complex of the DBR of BRCA1 421-701aa and splayed-arm DNA or the DBR alone, and N-terminus sequencing of 3 BRCA1 fragments cleaved from the DBR alone (A, B, C). The DNA-BRCA1 complex and BRCA1 alone were incubated at room temperature for 12 days. The proteins were separated by SDS-PAGE, electroblotted to PDVF membranes, and the bands on the membrane were excised for N-terminus sequencing to identify the N-terminal sequence of the cleaved fragments. (F) N-and C-terminus of DBR (421-701aa) binds to dsDNA in EMSA. The identities of the samples are shown on the figure. The concentration of labeled dsDNA and proteins are 40nM and 100nM, respectively. (G) BRCA1 binds dsDNA in a sequence-independent manner in EMSA. The sequences of 3 dsDNA with random sequences are listed in Table S1. The concentration of labeled dsDNA and proteins are 40nM and 0, 100 or 200nM, respectively. (H) The binding affinity of BRCA1 421-701aa to various lengths of dsDNA in EMSA. The concentration of labeled dsDNA and proteins are 40nM and 100nM, respectively. (I) The image of binding of BRCA1 421-988aa or 281-701aa to plasmid DNA by atomic force microscopy. The white dots are the protein and the red structures are plasmid DNA. (J) A model of BRCA1 binding to dsDNA.