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. 2023 Feb 25;51(6):2725–2739. doi: 10.1093/nar/gkad096

Figure 4.

Figure 4.

Activation of the MutLα endonuclease must overcome DNA supercoiling. (A) MutLα endonuclease activity on supercoiled, closed circular pUC18 DNA substrate is inhibited in the presence of ethidium bromide (EtBr) intercalator. MutLα is included in reactions at 25, 50, 100, 150 and 200 nM final concentrations. RFC and PCNA are included at 100 nM and 500 nM, respectively along with 2.5 mM MnSO4 and 0.25 mM ATP. Where included, ethidium bromide is present at 5 μg/ml. Experiments were resolved by agarose gel and quantified data is reported here for reactions with (green squares) and without (black circles) ethidium bromide (EtBr). Number of replicates are 4. Data points represent averages and error bars indicate standard deviation between experiments. Graph was fit to hyperbolic model. (B) MutLα endonuclease activity on 2.7 kb pre-nicked DNA substrate (Nt.BspQI) in the presence (green squares) and absence (black circles) of ethidium bromide intercalator. The experiment is identical to that in panel A. Data points represent the average of three replicates. (C) MutLα endonuclease activity on 2.7 kb linear substrate generated by HindIII digestion in the presence (green squares) and absence (black circles) of ethidium bromide intercalator. The experiment is identical to that in panels A and B. Data points represent the average of three replicates.