title: Single-stranded DNA Binding by the Helix-Hairpin-Helix Domain of XPF Protein Contributes to the Substrate Specificity of the ERCC1-XPF Protein Complex.
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 andin vitroDNA 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.
authors.affiliation: From the Bijvoet Center for Biomolecular Research, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands and.
authors.forename: Devashish
authors.initials: D
authors.lastname: Das
authors.affiliation: From the Bijvoet Center for Biomolecular Research, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands and.
authors.forename: Maryam
authors.initials: M
authors.lastname: Faridounnia
authors.affiliation: the Department of Molecular and Biomedical Sciences, Jožef Stefan Institute, Jamova cesta 39, 1000 Ljubljana, Slovenia.
authors.forename: Lidija
authors.initials: L
authors.lastname: Kovacic
authors.affiliation: From the Bijvoet Center for Biomolecular Research, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands and.
authors.forename: Robert
authors.initials: R
authors.lastname: Kaptein
authors.affiliation: From the Bijvoet Center for Biomolecular Research, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands and.
authors.forename: Rolf
authors.initials: R
authors.lastname: Boelens
authors.affiliation: From the Bijvoet Center for Biomolecular Research, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands and g.e.folkers@uu.nl.
authors.forename: Gert E
authors.initials: GE
authors.lastname: Folkers
mesh_terms: D001665:Binding Sites; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D019281:Dimerization; D004720:Endonucleases / Q000378:metabolism*; D018257:Helix-Loop-Helix Motifs; D006801:Humans; D011485:Protein Binding; D013379:Substrate Specificity
chemical_list: D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; C095888:xeroderma pigmentosum group F protein; C071447:ERCC1 protein, human; D004720:Endonucleases
keywords: DNA repair; DNA-protein interaction; ERCC1-XPF; NMR; XPF homodimer; helix-hairpin-helix domain; nucleotide excision repair; single-stranded DNA binding; structure-function