Figure 5.

SLX4 Promotes Unhooking of an ICL by XPF-ERCC1

(A) Outline of forked substrate containing a single nitrogen mustard-like interstrand crosslink (ICL) and its predicted reaction products. The substrate was generated from two oligonucleotides (each 35 nucleotides in length) with a crosslink between adjacent guanines, close to the ss/ds junction (red boxed inset). Sequential unhooking of the crosslinked DNA should result in an intermediate product (≫35 nt), followed by final products >35 and ≤15 nt (illustrated with green and red arrowheads).

(B) The forked ICL substrate or an identical, but noncrosslinked, control (YF) were radiolabeled at the 5′ end and reacted with XE or SXE enzyme complexes and analyzed by denaturing PAGE. Cleavage sites and reaction products corresponding to those illustrated in (A) are shown as arrows and brackets (the equivalent products from YF migrate at 19 and 15 nt). Comparison of ICL and YF digestion reveals the first ≫35 nt product is most likely to result from an incision at the ss/ds boundary (corresponding to 19 nt product of noncrosslinked YF fork). This was confirmed with 3′-end labeling (Figure S5).

(C) The primary reaction product (≫35 nt, green box) from the ICL substrate was purified as a substrate in a second reaction (“incised ICL”) to test whether the ICL was cleaved again (unhooked). The 15 and >35 nt product (red arrowhead and brackets) correspond to cleavage 5′ of the adducted guanine. All reactions contained 5 nM enzyme complex and ∼1.5 pM substrate. An asterisk denotes a low abundance, background band (a contaminant noncrosslinked oligonucleotide). Representative gels depict experiments performed at least three times.