(A) The gel image shows EXO1 activity on flap substrates. Lanes 1 and 2 contain a 5′ label DNA substrate GK247 in absence and presence of EXO1 (star denotes 5′-radiolabelling site). Lane 3 contains 5′ label 10 nt oligo (GK245). (B) Schematic overview of EXO1 processing hairpin forked DNA substrate. The forked substrate with a 10 nt flap is cleaved 1 bp inwards at the dsDNA resulting in 11 nt and smaller product of 9 nt (indicated with grey arrow). The 1 nt cleavage position at the ssDNA is indicated with a red arrow. (C) EXO1 process short flaps efficiently. Representative gel of five independent biological repeats of EXO1 showing nuclease activities on 5′-flap DNA substrates in length between 1 and 20 nt 5′ flap (star denotes 5′-radiolabelling site). DNA oligos GK251–GK253 are in range of 1 nt–5 nt 5′ flap and GK246 and GK249 contain respectively 9 nt and 20 nt 5′-flap. The different DNA substrates are setup in absence and presence of EXO1. (D) Analysis of the distribution of substrate and degraded flap and 1 nt product. (E) Statistical analysis of the degradation of the flap substrates. One-way analysis of variance with Tukey's multiple-comparison test of the EXO1 nuclease activity in salt condition. Percent of EXO1 activity on different 5′-flap DNA substrates. The 1 and 2 nt DNA 5′-flap are both significantly (***P<0.001) more degraded than longer flaps (>2 nt).