Figure 6: The stimulation of RecN ATPase by RecA protein under D-loop assay conditions is target DNA concentration- and length-dependent. | Nature Communications

Figure 6: The stimulation of RecN ATPase by RecA protein under D-loop assay conditions is target DNA concentration- and length-dependent.

From: The cohesin-like RecN protein stimulates RecA-mediated recombinational repair of DNA double-strand breaks

Figure 6

Reactions were carried out in Buffer A and assembled as in reaction 6, including protein concentrations, from Fig. 5b, except as noted. (a) The steady-state RecN ATP hydrolysis rate was measured when added to RecA K83R protein incubated with probe DNA (where indicated) followed by target DNA (where indicated). The probe DNA concentration is held constant at 10 μM and the target DNA concentration is titrated (0–10 μM), and white bars represent the rate of ATP hydrolysis catalysed by RecN protein at each DNA concentration relative to the rate measured at zero target DNA. The target DNA concentration is held constant at 10 μM and the probe DNA concentration is titrated (0–10 μM), and grey bars represent the rate of ATP hydrolysis catalysed by RecN protein at each DNA concentration relative to the rate measured at zero probe DNA. Error bars represent the s.d. of the relative rate (the s.d. of the average rate divided by the average rate) from 3 to 15 independent experiments (see Table 2 for steady-state rates). (b) The steady-state RecN ATP hydrolysis rate was measured when added to RecA K83R protein incubated with 2 nM molecules (10 μM nt) probe DNA followed by 2 nM molecules linearized, target DNA (target lds DNA, black square) or 10 μM of nucleotides linearized, target DNA (target lds DNA, white square) of different lengths, as indicated. The linear duplex target DNA substrate length in kilobase pairs (kbp) and concentration in μM nt and nM molecules for the two sets are as follows: 0.65 kbp (2.6 μM, black square; 7.7 nM, white square); 1.1 kbp (4.3 μM, black square); 1.5 kbp (5.8 μM, black square; 3.5 nM, white square); 2.4 kbp (9.6 μM, black square; 2.1 nM, white square); 4.2 kbp (16.8 μM, black square); 5.4 bp (21.5 μM, black square; 0.9 nM, white square); and 8.7 kbp (34.8 μM, black square; 0.6 nM, white square). The error bars represent the s.d. of four independent experiments. nt, nucleotide.

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