Figure 6.

SLX4 Increases the Efficiency of XPF-ERCC1 ICL Unhooking

(A) WT SXE or SXE harboring either XPF D678A or R690S mutations were incubated with the ICL substrate labeled at the 5′ end. ICL cleavage products are clearly seen with WT SXE, whereas SXE R690S (associated with human FA) shows very weak activity. The cleavage products are illustrated with a green arrow, red bracket, and red arrow (as described in Figure 5). A noncrosslinked oligonucleotide contaminant is marked with an asterisk.

(B) Representative time course, comparing reaction of ICL substrate with either XE or SXE.

(C) Rates of substrate turnover for ICL or equivalent noncrosslinked control, calculated from data presented in (B).

(D) Graph representing the ICL product formation for XE (blue) and SXE (red) enzyme complexes. Filled symbols mark the first incision product (shown in B above as a green arrow), open symbols depict 15 nt product (B, red arrow). The accumulation of the 15 nt product is dependent on the first product and marks the “unhooking” of the crosslink. Assays were performed with 5 nM enzyme complex and ∼1.5 pM labeled substrate, incubated for the time indicated, quenched, and separated by 12% denaturing PAGE gel. Data in (C) and (D) are plotted from a minimum of three independent experiments; error bars represent SEM.