Biophysical Characterization of the Interaction Domains and Mapping of the Contact Residues in the XPF-ERCC1 Complex

Abstract

XPF and ERCC1 exist as a heterodimer to be stable and active in cells and catalyze DNA cleavage on the 5-side of a lesion during nucleotide excision repair. To characterize the specific interaction between XPF and ERCC1, we expressed the human ERCC1 binding domain of XPF (XPF-EB) and the XPF binding domain of ERCC1 (ERCC1-FB) in Escherichia coli. Milligram quantities of a heterodimer were characterized with gel filtration chromatography, an Ni 2؉ -NTA binding assay, and analytical ultracentrifugation. Cross-linking experiments at high salt concentrations revealed that XPF interacts with ERCC1 mainly through hydrophobic interactions. XPF-EB was also shown to homodimerize in the absence of ERCC1. NMR cross-saturation methods were applied to map the residues involved in formation of the XPF-EB⅐XPF-EB homodimer and the XPF-EB⅐ERCC1-FB heterodimer. Helix H3 and the C-terminal region of XPF-EB were either within or in close proximity to the homodimer interface, whereas the ERCC1-FB binding site of XPF-EB was distributed across helix H1, a small part of H2, H3, and the C-terminal region, most of which exhibited large changes in chemical shift upon ERCC1 binding. The XPF-EB heterodimeric interface is larger than the XPF-EB homodimeric one, which could explain why XPF has a stronger affinity for ERCC1 than for a second molecule of XPF. The XPF binding sites of ERCC1 were located in helices H1 and H3 and in the C-terminal region, similar to the involved surface of XPF. We used cross-saturation data and the crystal structure of related proteins to model the two complexes.DNA lesions, which interfere with DNA replication, transcription, and recombination (1), occur as a result of exogenous and endogenous damaging agents, such as UV irradiation, products of oxidative stress, and DNA-reactive chemicals. To maintain the integrity of the genome, DNA lesions are efficiently removed by DNA repair pathways, such as the nucleotide excision repair pathway, which removes various types of bulky DNA adducts. During nucleotide excision repair, the 5Ј and 3Ј sides of a lesion are cut by two endonucleases, the XPF-ERCC1 heterodimeric complex and XPG, respectively (2-5). This dual incision is made asymmetrically around a lesion to allow its release as part of a larger DNA fragment (24 -32 nucleotides) (6 -8). The remaining gap is filled by DNA synthesis and ligation (9, 10).The XPF-ERCC1 complex cuts a variety of DNA substrates, including bubbles, stem-loops, splayed arms, and flaps (2,4,11,12). XPF contains the endonuclease activity, while ERCC1 likely modulates the specificity required for DNA incision (13). XPF and ERCC1 form a stable heterodimer in mammalian cells and in vitro, and various lines of evidence suggest that both ERCC1 and XPF are unstable in the absence of the respective partner of each (14 -18). The XPF-ERCC1 heterodimer is known to be formed through the interaction of their C-terminal domains, which each include two helix-hairpin-helix (HhH) 1 motifs (19 -21). However, a detailed characterization of the XPF-ERCC1 heterod...

Mus81-Mms4 Functions as a Single Heterodimer To Cleave Nicked Intermediates in Recombinational DNA Repair

“…Another possibility is that the interactions observed under overexpressed conditions may not accurately reflect what occurs at physiological protein levels. Other XPF endonuclease family members have been shown to artificially associate when overexpressed or when their obligate partners are not present (15,18,25). Our analysis of the association of the Mus81 and Mms4 proteins at their endogenous levels eliminates any concern about artificial dimerization and provides a clearer depiction of what is occurring in vivo.…”

Emodin enhances cisplatin-induced cytotoxicity via down-regulation of ERCC1 and inactivation of ERK1/2

“…Cisplatin interacts with DNA to form predominantly intra-strand cross-linked DNA adducts that trigger nucleotide excision repair machinery or induce cell death [32][33][34]. When ERCC1 combines with XPF endonuclease (xeroderma pigmentosum complementation group F, ERCC4) [35,36], it can act as a heterodimer and function as an endonuclease to catalyze incision on the 5 -side of the damaged DNA [37]. ERCC1 plays an important role in repairing double-strand breaks induced by DNA inter-strand cross-links.…”

DNA repair gets physical: Mapping an XPA-binding site on ERCC1

“…In addition, the same domain was crystallized from the archaea A. pernix XPF in the presence and absence of DNA [17]. The all α-helix structure is the dimerization domain between ERCC1 and XPF and it revealed an extensive network of hydrophobic interactions that are shared between the two proteins [15,17,29]. Thus explaining how the two proteins mutually stabilize one another.…”

“…ERCC1 also contains a Cterminal HhH 2 fold which is essential for dimerization with XPF [16]. ERCC1 and XPF mutually stabilize one another through interactions between their HhH 2 domains [16,[26][27][28][29]. ERCC1's nuclease fold has been solved by NMR and X-ray crystallography ( [1,2,15], PDB records 2JPD, 2JNW and 2A1I, respectively) and the nuclease domain of Aeropyrum pernix XPF-like protein has been solved with and without DNA ( [17], PDB files 2BHN, 2BGW).…”