Structure and Stability of ERCC1-XPF DNA Repair Complexes

Abstract

Understanding DNA repair pathways such as Nucleotide Excision Repair, Double Strand Break repair and Interstrand Cross-Link repair is of basic interest for understanding fundamental cellular processes. It also forms the basis for understanding molecular details of diseases when defects occur in these pathways. Additional insight will help us to define relations to other cellular processes, and to find relations between the different DNA repair pathways, ageing and cancer. The ERCC1-XPF structure-specific endonuclease complex, as a member of multiple DNA repair pathways, is responsible for the incision of DNA upstream of the DNA damage site. Several symptoms, ranging from mild to severe, are related to mutations in XPF and/or ERCC1. Moreover, the involvement of ERCC1-XPF in DNA repair, especially the removal of damages induced during platinum-chemotherapy in various cancer types, results in tumor resistance to these treatments. This reflects another horizon in medical importance of ERCC1-XPF, and has increased the attention to possible regulation of ERCC1-XPF expression and activity. In fact, the correlation between ERCC1 expression and the resistance to chemotherapeutic treatments makes ERCC1 a potential biomarker for the prediction of chemoresistance and patient survival. In the research described in this thesis the heterodimeric complex of the tandem Helix-hirpin-Helix (HhH)2 domains of ERCC1-XPF was studied, as well as its stability and role in DNA recognition. In her thesis, Maryam Faridounnia, has given a detailed look into the interaction between ERCC1 and XPF in the ERCC1-XPF complex. She has analyzed the stability of the ERCC1-XPF complex from a thermodynamic and a kinetic perspective and has discussed the factors contributing to the stability of the heterodimeric complex. Furthermore, she presented a model that explains the preferred formation of ERCC1-XPF heterodimers. Then, she investigated the effects of the disease-related F231L ERCC1 mutation (F231L). F231 is part of the cavity of ERCC1, which contains the F894 anchor of XPF. It is shown that the F231L mutation causes a small but significant destabilization of the interaction between ERCC1 and XPF. Loss of function of ERCC1-XPF endonuclease in diverse DNA repair pathways especially ICL repair can in some cases lead to the severe COFS syndrome. In principle, this explains the observed phenotype of the patient for which the F231L mutation was established. A functional property of the ERCC1-XPF (HhH)2 domains is its capacity to bind near damaged DNA to the single strand/double strand (ds/ss)DNA junctions. Maryam examined the model for binding of ERCC1-XPF at a ds/ssDNA junction. This model was initially based on interactions between XPF (HhH)2 homodimers and short ssDNA nucleotides. The original model is validated with interaction studies of heterodimeric ERCC1-XPF and different DNA substrates, including ds/ssDNA forks. The biochemical and biophysical data show that the ERCC1 (HhH)2domain binds primarily to dsDNA and that at the same time the XPF (HhH)2 domain can bind to ssDNA. Therefore, the heterodimeric ERCC1-XPF complex can optimally bind at a ds/ssDNA junction as present during NER DNA repair.