Fig 2: MCPH1 stabilizes RAD51 and DMC1 nucleoprotein filaments. (A) Schematic of our Benzonase protection assay. (B) The 5'-32P-labeled 80-mer ssDNA (1.5 µM nucleotides) was pre-incubated with RAD51 and then incubated with tag-free MCPH1 (lanes 5–7). RAD51 (lane 3) or MCPH1 alone (lane 4) were included as negative controls. RAD51 with AMP-PNP was included as a positive control. Then, Benzonase was added to challenge the filaments. The reactions were deproteinized and electrophoresed in 10% TBE polyacrylamide gels. The gels were dried and analyzed by phosphorimaging. The 32P-label is denoted by the asterisk. (C, D) The assay was also conducted using DMC1 (C) or RecA (D) and analyzed in the same way. DMC1 with calcium (0.5 mM) or RecA with ATP?S were included as respective positive controls. Plots of percentage DNA protected are shown below the gels. Error bars represent the standard deviation (±SD) calculated from three independent experiments.

Fig 3: MCPH1 physically interacts with RAD51 and DMC1. In an affinity pulldown assay, purified (His)6-MBP-MCPH1 was pre-incubated with RAD51 (A), DMC1 (B) or RecA (C). The mixtures were then further incubated with Talon resin to capture protein complex via the (His)6-tag. The supernatant (S), wash (W), and eluate (E) from the reactions were analyzed by means of 12% SDS-denaturing polyacrylamide gels with Coomassie Blue staining. RAD51, DMC1 and RecA alone were included as controls (panels A, B and C; lanes 1–3). (D) Amylose resin was used to pull-down MBP proteins, with RAD51, DMC1 and RecA as negative controls.