Gel from Scientific Research

MutSβ not MutSα inhibits FAN1. Protein-free undigested slip-out DNA substrate (100 nM), lane 1. Slip-outs were preincubated with buffer, lane 2, or increasing concentrations of purified human MutSα (50, 100 or 200 nM), lanes 3–5; or MutSβ (50, 100 or 200 nM), lanes 6–8. Nuclease digestions were initiated by addition purified human FAN1 (50 nM). Lanes 9 and 10 have slip-out DNA and only MutSα (50 nM) or only MutSβ (50 nM), ensuring these purified proteins are nuclease-free. For gels in panels a and b, the percentage cleavage for each reaction was normalized to cleavage levels with FAN1 alone (lane 2), and these levels were graphed (GraphPad prism 9.1). The vertical schematic to the right of each gel indicates the location of cleavage sites along the FAM-labeled DNA strand. ‘E’ is elbow at the dsDNA-ssDNA junction, blue arrowheads represent cleavage hotspots. Results of two-sided unpaired t -test are indicated (versus FAN1 alone in panel b, or versus ‘no FAN1’ in panels c and d). n = 2 experiments for panel a, n = 3 experiments for the other panels and mean ± standard deviation (s.d.) are plotted.

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