NucS-DNA dissociation at 23°C.

<p>a) Cumulative proportion of observation time for: (i) photobleaching of NucS-Alexa 488 immobilized on the coverslip in the absence of DNA (blue plain line) fitted by an exponential function (purple dashed line: with <i>t_c</i>  = 2.0 s), (ii) NucS interacting non-specifically with a treated surface (red dashed line) and (iii) NucS interacting with 20-bp long 3′-flaps in 150 mM NaCl buffer (green plain line). The corresponding histogram of raw data for case (iii) is shown in the inset. ***: <i>p<</i>10<i>⁻</i>³. Note that this graph presents the raw residence time distribution and consequently events used to construct the green curve are a mix of photobleaching and dissociation. All the other results presented in this figure (b–d) are dissociation time distributions obtained from the raw distributions as mentioned in the main text. b) NucS cumulative interaction time distribution on 3′-flaps in 150 mM NaCl buffer (green squares) with an exponential fit <i>k_off</i>  = 0.076 s⁻¹(red line). The blue dashed lines are exponential functions using the limit values defined by the standard error (<i>k_off</i>  = 0.069 s⁻¹ and <i>k_off</i>  = 0.082 s⁻¹). c) Interaction time distribution at 50 mM (left) and 150 mM (right) salt for 5′-flaps (blue squares) and 3′-flaps (green circles). d) Left: interaction time distribution for 3′-flaps at different glycerol concentration (blue dashed line: 5%, purple dashed line: 20%) Right: interaction time distribution for 3′-flaps of different length (blue dashed line: 20 bp, red dashed line: 30 bp). On panels c) and d), we present the cumulative distribution of interaction time instead of their distribution to perform comparisons without biases due to the construction of histograms.</p

NucS-DNA flap association at 23°C.

<p>a) Total number of detected interactions as a function of time (green squares) together with a linear fit (red dashed line <i>N</i> = 0.13<i>t</i>) yielding a constant effective on-rate. b) <i>k_on</i> measured for different substrates at 150 mM NaCl either for wild-type NucS (WT) or a binding-deficient mutant (R70A). 3′-flaps (green), 5′-flaps (blue), 5′flaps with Dig-AntiDig blocked extremity (pink) and dsDNA (red). c) Association rate constant <i>k_on</i> measured for 3′-flaps (green) and 5′-flaps (blue) at different salt concentrations (50 mM and 150 mM NaCl). d) Association rate constant <i>k_on</i> measured for 3′-flaps for different glycerol concentrations (0%, 5% and 20%).</p

NucS-DNA flap association rate constants for different salt and glycerol values for the 3′ flap.

<p>NucS-DNA flap association rate constants for different salt and glycerol values for the 3′ flap.</p

Dissociation rates of NucS on DNA flaps and dissociation constants .

<p>Dissociation rates of NucS on DNA flaps and dissociation constants .</p

Association rate constants at 45°C.

<p>Association rate constants at 45°C.</p

Dissociation rates at 45°C.

<p>Dissociation rates at 45°C.</p