NMR study on XPF-ERCC1 complex

Abstract

XPF-ERCC1 is a heterodimeric protein complex involved in nucleotide excision repair(NER), DNA cross-link repair and recombination. XPF-ERCC1 acts as a structure-specific DNA endonuclease, cleaving at duplex-single-stranded DNA junctions. In repair, XPF-ERCC1 makes an incision on the 5’ side of the lesion. XPF or ERCC1 alone is unstable in mammalian cells in the absence of its partner. To understand the reason why XPF and ERCC1 are unstable without partners, the structural studies of the interaction domains are needed. We expressed the recombinant proteins (ERCC1 binding domain of XPF and XPF binding domain of ERCC1), in order to obtain large amount of XPF-ERCC1 complex for the structural analysis. The purified XPF sub-domain was present as a soluble trimer, which was confirmed by gel permeation chromatography (GPC). However, ERCC1 sub-domain was expressed as an inclusion body and it was solublized with 8M urea and purified using Sepharose column under denaturing condition. The refolding of XPF and ERCC1 together was achieved via the stepwise dialysis to remove the denaturant. We obtained milligram quantities of XPF-ERCC1 complex from the optimized refolding protocol . XPF-ERCC1 complex formation is established by a direct interaction between these two binding domains. Refolded XPF-ERCC1 complex is native state as shown by GPC, NMR and binding study. The complex formation as a soluble form is essential to structural studies of the interaction domains. NMR studies of these domains can be used to localize protein-protein binding site and to obtain detailed structures of these domains. These structural studies can provide critical information on interaction between XPF sub-domain and ERCC1 sub-domain. This may suggest why the proper complex formation is required for stability of the two proteins.