Extended Data Fig. 6: Characterization of PARP1 interaction with BRCA2 and RAD51.
From: BRCA2 prevents PARPi-mediated PARP1 retention to protect RAD51 filaments
(a) Control western blots showing bead-capture experiment to measure binding of RAD51 to ss/dsDNA co-incubated with PARP1 ± NAD+ ± PARPi. (b) Coomassie stain and western blot for human XRCC1 protein highlighting the quality of the protein used for the protein pull-down assays. (c) First and last washes from the pulldown assays performed in Fig. 3d. (d) Protocol for Protein-A Magnetic Beads pull-down assays for untagged PARP1 with XRCC1, BRCA2 and RAD51 in absence and presence of PARPi. (e) Protein A Magnetic Beads pull-down assay without PARPi. Lane 1: Marker, lane 2: “Control” no IgG1 against PARP1, lane 3: IgG1 against PARP1 + recombinant PARP1 (0.1 µM), lane 4-5: IgG1 for PARP1 + recombinant PARP1 (0.1 µM) ± NAD+ (0.25 µM) with XRCC1/2X MBP-BRCA2/RAD51 (0.1 µM each). (f) Protein A Magnetic Beads pull-down assay with PARPi. Lane 1: Marker, lane 2: “Control” no IgG1 against PARP1, lane 3–5: IgG1 against PARP1 + NAD+ + PARPi with XRCC1/2XMBP-BRCA2/RAD51. (g) Control experiment demonstrating addition of NAD+ or PARPi does not affect BRCA2-RAD51 mediated strand exchange reaction (Lane5,6). Lanes 1–4 are also shown in Fig. 1c. (h) TDP generated from HMM analysis of the smFRET trajectories for PARP1 + NAD+ + PARPi bound to 3′ tailed DNA followed by addition of RAD51 highlighting the transition densities corresponding to each state (Nucleation, N) for RAD51 and (PARP1 Retention, PR) for PARP1 respectively. Brighter colors represent more frequent transitions, and the frequency scale is shown to the right of the graphs. (i) Dwell time histograms (bin size = 90 msec) for each of the FRET states as obtained from HMM analysis along with the FRET population histograms for unprocessed and unnormalized raw data from ebFRET output (Supplementary Table 1) (n = 632; r = 2). The number of analyzed molecules and replicates are denoted by n and r, respectively. [BRCA2] = 0.04 µM | [RAD51] = 0.4 µM | [ATP] = 2 mM. [PARP1] = 0.4 µM | NAD+ = 1 mM | [PARPi] = 0.1 µM talazoparib (BMN673).
