The nucleotide excision repair protein complex ERCC1-XPF is required for incision of DNA upstream of the DNA damage. Functional studies have provided insight into the binding of ERCC1-XPF to various DNA substrates. However, since no structure for the ERCC1-XPF-DNA complex has been determined, the mechanism of substrate recognition remains elusive. Here we biochemically characterize the substrate preferences of the Helix-hairpin-Helix domains of XPF and ERCC-XPF and show that the binding to ss/dsDNA junctions is dependent on joint binding to the DNA binding domain of ERCC1 and XPF. We reveal that the homodimeric XPF is able to bind various ssDNA sequences, but with a clear preference for guanine containing substrates. NMR titration experiments and in vitro DNA binding assays show that also within the heterodimeric ERCC1-XPF complex XPF specifically recognizes ssDNA. On the other hand, the HhH domain of ERCC1 preferentially binds dsDNA through the hairpin region. The two separate non-overlapping DNA binding domains in the ERCC1-XPF heterodimer jointly bind to an ss/dsDNA substrate and thereby, at least partially, dictate the incision position during damage removal. Based on structural models, NMR titrations, DNA-binding studies, site-directed mutagenesis, charge distribution and sequence conservation, we propose that the HhH domain of ERCC1 binds to dsDNA upstream of the damage and XPF binds to the non-damaged strand within a repair bubble.
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