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. 2014 Feb 3;33(5):482–500. doi: 10.1002/embj.201386100

Figure 4.

Figure 4

Rad50 catalytic domain mutations affect ATP-dependent DNA binding and hydrolysis.
  1. ATP binding by full-length MR complexes as measured by quenching of tryptophan fluorescence by TNP-ATP. Shown are emission scans using 290 nm excitation and TNP-ATP as indicated.
  2. Quantitation of quenching efficiency (F0/F) at 330 nm emission calculated from (A).
  3. ATPase kinetics measurements of Rad50cd-linked and full-length MR proteins (see also Supplementary Fig S4).
  4. Gel-shift assays of Rad50cd-linked (2.5 μM) with a 41-bp DNA duplex substrate.
  5. Gel-shift assays of MRcd complexes (400 nM) with a 41-bp DNA duplex substrate. Protein-DNA complexes are indicated (I and II), with the upper band (I) being nucleotide-dependent.
  6. Gel-shift assays as in (E) with full-length MR (200 nM).
  7. Crystal structures reveal hydrogen-bonding rearrangements between nucleotide-free WT (left) and R805E Rad50cd-linked (right). Colored as in Fig C, with the K54 loop in burgundy and red arrows highlighting major changes. See also Supplementary Fig S4.

Source data are available online for this figure.