Figure 4.

Nuclease Activity of Altered XPF

(A) NER activity of wild-type and altered ERCC1-XPF dimer. A plasmid containing a site-specific 1,3-intrastrand cis-Pt DNA crosslink was incubated with whole-cell extracts from HeLa cells or XPF-deficient cells (XP2YO) complemented with recombinant ERCC1-XPF purified from Sf9 insect cells as reported.¹³ The excised DNA fragments of 24–32 nucleotides are shown. The position of a 25-mer is indicated.

(B) Incision of a stem-loop substrate with wild-type and altered XPF. The 3′ Cy5-labeled substrate was incubated with recombinant ERCC1-XPF in the presence of 2 mM MgCl₂ or 0.4 mM MnCl₂, and the products were analyzed by denaturing PAGE. The incision reaction was performed essentially as described earlier.^5,11