Single-stranded DNA Binding by the Helix-Hairpin-Helix Domain of XPF Protein Contributes to the Substrate Specificity of the ERCC1-XPF Protein Complex

Devashish Das,Maryam Faridounnia,Lidija Kovacic,Robert Kaptein,Rolf Boelens,Gert E Folkers

doi:10.1074/jbc.m116.747857

Devashish Das, Maryam Faridounnia + Show 4 more

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https://doi.org/10.1074/jbc.m116.747857

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Abstract

The nucleotide excision repair protein complex ERCC1-XPF is required for incision of DNA upstream of DNA damage. Functional studies have provided insights into the binding of ERCC1-XPF to various DNA substrates. However, because 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 (HhH) domains of XPF and ERCC-XPF and show that the binding to single-stranded DNA (ssDNA)/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 also show that, 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 ssDNA/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.

Highlights

The nucleotide excision repair protein complex ERCC1-XPF is required for incision of DNA upstream of DNA damage
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
Similar to full-length ERCC1-XPF heterodimers, these HhH domains can together form stable complexes with various ss/ds junction-containing DNA, like bubble, hairpin, and splayed arm substrates [32,33,34]. These findings suggest that structure-specific DNA binding by the ERCC1XPF heterodimer is dependent on the HhH domain regions of both proteins

Summary

Edited by Patrick Sung

The nucleotide excision repair protein complex ERCC1-XPF is required for incision of DNA upstream of DNA damage. Postnatal phenotypes of XPF and ERCC1 knockout mice suggest additional functions for ERCC1-XPF in double strand break repair [22], single strand annealing [23], interstrand cross-link repair [24, 25], telomere maintenance [26, 27], and gene-targeting events [28] All of these genome regulatory processes require binding of ERCC1-XPF at distinct DNA sequences, involving various protein complexes (29 –31). We could show that besides nonspecific phosphate backbone contacts involving the second helix of the first HhH motif, a cavity is formed between the two motifs of the HhH domain, where a guanine base can be bound These observations led us to propose that, in contrast to the model proposed by Tsodikov et al [36], the ERCC1-XPF heterodimer recognizes DNA substrates involving the two individual DNAbinding surfaces present in ERCC1 and XPF that preferentially bind dsDNA and ssDNA, respectively [34]. The concerted binding of the HhH domains of ERCC1 and XPF to dsDNA and ssDNA, respectively, is essential for the correct positioning on the ssDNA/dsDNA junction

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