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. 2016 Oct 26;35(23):2584–2601. doi: 10.15252/embj.201593132

Figure EV7. MMS22L–TONSL promotes DNA strand exchange by RAD51.

Figure EV7

  • A–C
    Quantification of DNA strand exchange with RAD51, with or without MMS22L–TONSL (75 nM) using a 3′‐tailed (A), 5′‐tailed (B), or gapped (C) DNA substrate. Averages are shown, n = 2; error bars, SEM.
  • D
    A representative DNA strand exchange experiment with ssDNA and RAD51, as indicated with or without ATP. See scheme in Fig 6A.
  • E
    A representative DNA strand exchange experiment with ssDNA, RAD51, MMS22L–TONSL (MT), as indicated with or without ATP. See scheme in Fig 6A.
  • F, G
    Representative DNA strand exchange experiments with a fixed concentration of RAD51 (270 nM) and varying concentrations of MMS22L–TONSL (F) or MMS22L (G). See scheme in Fig 6A.
  • H, I
    Representative electrophoretic mobility shift assays with MMS22L–TONSL, MMS22L, RAD51, and either dsDNA (H) or ssDNA (I). Note that MMS22L–TONSL reduces RAD51 binding to dsDNA, but does not affect binding of RAD51 to ssDNA.
  • J
    A representative DNA strand exchange experiment with RAD51 (120 nM), ssDNA, RPA (30 nM), and MMS22L–TONSL. Note that MMS22L–TONSL is not capable to alleviate the inhibitory effect of RPA on DNA strand exchange.
  • K
    MMS22L (50 nM), MMS22L–TONSL (50 nM), RAD51 (500 nM), and RPA (100 nM) were incubated with biotinylated ssDNA in a buffer containing 20 mM Tris–HCl (pH 7.5), 0.1% Triton X‐100, 0.1 mg/ml bovine serum albumin, 120 mM NaCl, 2 mM CaCl2, 10 mM magnesium acetate, 1 mM dithiothreitol with or without ATP (2 mM). MMS22L–TONSL, but not MMS22L, forms a ternary complex with ssDNA‐bound RAD51 in the presence of ATP.
  • L
    Similar assay as in (K), but in a buffer lacking calcium, magnesium, and ATP. Under conditions that do not favor RAD51 filament formation, MMS22L–TONSL preferentially binds RPA‐coated ssDNA.