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

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

Structure and Stability of ERCC1-XPF DNA Repair Complexes

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

Structure and Stability of ERCC1-XPF DNA Repair Complexes

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

Prediction of Protein Quaternary Structures

Determination of the protein structure and understanding its function is essential for any relevant medical, engineering, or pharmaceutical applications. Therefore, the study of quaternary structure of proteins, despite all the obstacles in acquiring data from large macromolecular assemblies, is one of the major goals in biomolecular sciences. This chapter discusses respectively protein structure prediction, template-based predictions, critical assessment of protein structure prediction (CASP), and quaternary structure prediction. Homology modeling and threading methods are two types of template-based approaches. The homology modeling method needs to have the homologous protein structure as template and threading methods are a new approach in fold recognition, in which the tool attempts to fit the sequence in the known structures. A few sequence-based computational methods have been developed for the prediction of protein quaternary structure using statistical models or machine learning methods. © 2016 John Wiley & Sons, Inc

Prediction of Protein Quaternary Structures

Summary Determination of the protein structure and understanding its function is essential for any relevant medical, engineering, or pharmaceutical applications. Therefore, the study of quaternary structure of proteins, despite all the obstacles in acquiring data from large macromolecular assemblies, is one of the major goals in biomolecular sciences. This chapter discusses respectively protein structure prediction, template‐based predictions, critical assessment of protein structure prediction (CASP), and quaternary structure prediction. Homology modeling and threading methods are two types of template‐based approaches. The homology modeling method needs to have the homologous protein structure as template and threading methods are a new approach in fold recognition, in which the tool attempts to fit the sequence in the known structures. A few sequence‐based computational methods have been developed for the prediction of protein quaternary structure using statistical models or machine learning methods

Protein cold adaptation: Role of physico-chemical parameters in adaptation of proteins to low temperatures

During years 2007 and 2008, we published three papers (Jahandideh, 2007a, JTB, 246, 159-166; Jahandideh, 2007b, JTB, 248, 721-726; Jahandideh, 2008, JTB, 255, 113-118) investigating sequence and structural parameters in adaptation of proteins to low temperatures. Our studies revealed important features in cold-adaptation of proteins. Here, we calculate values of a new set of physico-chemical parameters and perform a comparative systematic analysis on a more comprehensive database of psychrophilic-mesophilic homologous protein pairs. Our obtained results confirm that psychrophilicity rules are not merely the inverse rules of thermostability; for instance, although contact order is reported as a key feature in thermostability, our results have shown no significant difference between contact orders of psychrophilic proteins compared to mesophilic proteins. We are optimistic that these findings would help future efforts to propose a strategy for designing cold-adapted proteins. © 2015 Elsevier Ltd

Prediction of Protein Quaternary Structures

Summary Determination of the protein structure and understanding its function is essential for any relevant medical, engineering, or pharmaceutical applications. Therefore, the study of quaternary structure of proteins, despite all the obstacles in acquiring data from large macromolecular assemblies, is one of the major goals in biomolecular sciences. This chapter discusses respectively protein structure prediction, template‐based predictions, critical assessment of protein structure prediction (CASP), and quaternary structure prediction. Homology modeling and threading methods are two types of template‐based approaches. The homology modeling method needs to have the homologous protein structure as template and threading methods are a new approach in fold recognition, in which the tool attempts to fit the sequence in the known structures. A few sequence‐based computational methods have been developed for the prediction of protein quaternary structure using statistical models or machine learning methods

The Cerebro-oculo-facio-skeletal Syndrome Point Mutation F231L in the ERCC1 DNA Repair Protein Causes Dissociation of the ERCC1-XPF Complex

The ERCC1-XPF heterodimer, a structure-specific DNA endonuclease, is best known for its function in the nucleotide excision repair (NER) pathway. The ERCC1 point mutation F231L, located at the hydrophobic interaction interface of ERCC1 (excision repair cross-complementation group 1) and XPF (xeroderma pigmentosum complementation group F), leads to severe NER pathway deficiencies. Here, we analyze biophysical properties and report the NMR structure of the complex of the C-terminal tandem helix-hairpin- helix domains of ERCC1-XPF that contains this mutation. The structures of wild type and the F231L mutant are very similar. The F231L mutation results in only a small disturbance of the ERCC1-XPF interface, where, in contrast to Phe(231), Leu(231) lacks interactions stabilizing the ERCC1-XPF complex. One of the two anchor points is severely distorted, and this results in a more dynamic complex, causing reduced stability and an increased dissociation rate of the mutant complex as compared with wild type. These data provide a biophysical explanation for the severe NER deficiencies caused by this mutation