Gel from Scientific Research

Increasing FAN1 concentration can overcome MutSβ-mediated inhibition of cleavage. Protein-free undigested slip-out (100 nM), lane 1. Slip-outs were preincubated with buffer, lanes 1–4 or with MutSβ (200 nM), lanes 5–7. Nuclease digestions were initiated by adding increasing amounts of FAN1 (50, 100 or 200 nM), lanes 2–4 and 5–7. Lane 8 has slip-out and only MutSβ (50 nM). 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). For gels in panels c and d, the percentage cleavage for each reaction were graphed. 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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