ERCC1-XPF Complex Research Articles

ERCC1 and ERCC2 Polymorphisms Predict the Efficacy and Toxicity of Platinum-Based Chemotherapy in Small Cell Lung Cancer.

Standard first-line chemotherapy in small cell lung cancer (SCLC) is based on the platinum plus etoposide combination. Despite a high objective response rate, responses are not durable and chemotherapy-induced toxicity may compromise treatment. Genetic variants in genes involved in the DNA-repair pathways and in etoposide metabolization could predict treatment efficacy and safety and help personalize platinum-based chemotherapy. Germline polymorphisms in XRCC1, ERCC1, ERCC2, ABCB1, ABCC3, UGT1A1 and GSTP1 genes were investigated in 145 patients with SCLC. The tumor expression of ERCC1 was determined using immunohistochemistry, and the tumor expression of ERCC1-XPF was determined via a proximity ligation assay. Survival analyses showed a statistically significant association between the ERCC1 rs11615 variant and median progression-free survival (PFS) in patients with limited-stage (LS) SCLC (multivariate: hazard ratio 3.25, [95% CI 1.38-7.70]; p = 0.007). Furthermore, we observed differences between the ERCC1-XPF complex and median PFS in LS-SCLC, although statistical significance was not reached (univariate: positive expression 10.8 [95% CI 4.09-17.55] months versus negative expression 13.3 [95% CI 7.32-19.31] months; p = 0.06). Safety analyses showed that the ERCC2 rs1799793 variant was significantly associated with the risk of grade ≥ 3 thrombocytopenia in the total cohort (multivariate: odds ratio 3.15, [95% CI 1.08-9.17]; p = 0.04). Our results provide evidence that ERCC1 and ERCC2 variants may predict the efficacy and safety of platinum-based chemotherapy in SCLC patients. LS-SCLC patients may benefit most from ERCC1 determination, but prospective studies are needed.

Unveiling Novel ERCC1-XPF Complex Inhibitors: Bridging the Gap from In Silico Exploration to Experimental Design.

Modifications in DNA repair pathways are recognized as prognostic markers and potential therapeutic targets in various cancers, including non-small cell lung cancer (NSCLC). Overexpression of ERCC1 correlates with poorer prognosis and response to platinum-based chemotherapy. As a result, there is a pressing need to discover new inhibitors of the ERCC1-XPF complex that can potentiate the efficacy of cisplatin in NSCLC. In this study, we developed a structure-based virtual screening strategy targeting the inhibition of ERCC1 and XPF interaction. Analysis of crystal structures and a library of small molecules known to act against the complex highlighted the pivotal role of Phe293 (ERCC1) in maintaining complex stability. This residue was chosen as the primary binding site for virtual screening. Using an optimized docking protocol, we screened compounds from various databases, ultimately identifying more than one hundred potential inhibitors. Their capability to amplify cisplatin-induced cytotoxicity was assessed in NSCLC H1299 cells, which exhibited the highest ERCC1 expression of all the cell lines tested. Of these, 22 compounds emerged as promising enhancers of cisplatin efficacy. Our results underscore the value of pinpointing crucial molecular characteristics in the pursuit of novel modulators of the ERCC1-XPF interaction, which could be combined with cisplatin to treat NSCLC more effectively.

Design, synthesis and in vitro cell-free/cell-based biological evaluations of novel ERCC1-XPF inhibitors targeting DNA repair pathway

The structure-specific ERCC1-XPF endonuclease is essential for repairing bulky DNA lesions and helix distortions induced by UV radiation, which forms cyclobutane pyrimidine dimers (CPDs), or chemicals that crosslink DNA strands such as cyclophosphamide and platinum-based chemotherapeutic agents. Inhibition of the ERCC1-XPF endonuclease activity has been shown to sensitize cancer cells to these chemotherapeutic agents. In this study, we have conducted a structure activity relationship analysis based around the previously identified hit compound, 4-((6-chloro-2-methoxyacridin-9-yl)amino)-2-((4-methylpiperazin1-yl)methyl)phenol (F06), as a reference compound. Three different series of compounds have been rationally designed and successfully synthesized through various modifications on three different sites of F06 based on the corresponding suggestions of the previous pharmacophore model. The in vitro screening results revealed that 2-chloro-9-((3-((4-(2-(dimethylamino)ethyl)piperazin-1-yl)methyl)-4-hydroxyphenyl)amino)acridin-2-ol (B9) has a potent inhibitory effect on the ERCC1-XPF activity (IC50 = 0.49 μM), showing 3-fold improvement in inhibition activity compared to F06. In addition, B9 not only displayed better binding affinity to the ERCC1-XPF complex but also had the capacity to potentiate the cytotoxicity effect of UV radiation and inhibiting the nucleotide excision repair, by the inhibition of removal of CPDs, and cyclophosphamide toxicity to colorectal cancer cells.

ERCC1-XPF cooperates with CTCF and cohesin tofacilitate the developmental silencing of imprintedgenes.

Inborn defects in DNA repair are associated with complex developmental disorders whose causal mechanisms are poorly understood. Using an in vivo biotinylation tagging approach in mice, we show that the nucleotide excision repair (NER) structure-specific endonuclease ERCC1-XPF complex interacts with the insulator binding protein CTCF, the cohesin subunits SMC1A and SMC3 and with MBD2; the factors co-localize with ATRX at the promoters and control regions (ICRs) of imprinted genes during postnatal hepatic development. Loss of Ercc1 or exposure to MMC triggers the localization of CTCF to heterochromatin, the dissociation of the CTCF-cohesin complex and ATRX from promoters and ICRs, altered histone marks andthe aberrant developmental expression of imprinted genes without altering DNA methylation. We propose that ERCC1-XPF cooperates with CTCF and cohesin to facilitate the developmental silencing of imprinted genes and that persistent DNA damage triggers chromatin changes that affect gene expression programs associated with NER disorders.

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

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.

Quantification of the mutagenic potency and repair of glycidol-induced DNA lesions

Glycidol (Gly) is an electrophilic low-molecular weight epoxide that is classified by IARC as probably carcinogenic to humans. Humans might be exposed to Gly from food, e.g. refined vegetable oils, where Gly has been found as a food process contaminant. It is therefore important to investigate and quantify the genotoxicity of Gly as a primary step towards cancer risk assessment of the human exposure.Here, quantification of the mutagenic potency expressed per dose (AUC: area under the concentration-time curve) of Gly has been performed in Chinese hamster ovary (CHO) cells, using the HPRT assay. The dose of Gly was estimated in the cell exposure medium by trapping Gly with a strong nucleophile, cob(I)alamin, to form stable cobalamin adducts for analysis by LC–MS/MS. Gly was stable in the exposure medium during the time for cell treatment, and thus the dose in vitro is the initial concentration×cell treatment time. Gly induced mutations in the hprt-gene at a rate of 0.08±0.01 mutations/105 cells/mMh. Through comparison with the effect of ionizing radiation in the same system a relative mutagenic potency of 9.5rad-eq./mMh was obtained, which could be used for comparison of genotoxicity of chemicals and between test systems and also in procedures for quantitative cancer risk assessment.Gly was shown to induce strand breaks, that were repaired by base excision repair. Furthermore, Gly-induced lesions, present during replication, were found to delay the replication fork elongation. From experiments with repair deficient cells, homologous recombination repair and the ERCC1-XPF complex were indicated to be recruited to support in the repair of the damage related to the stalled replication elongation. The type of DNA damage responsible for the mutagenic effect of Gly could not be concluded from the present study.

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.

Molecular Docking and Molecular Dynamics Study on the Effect of ERCC1 Deleterious Polymorphisms in ERCC1-XPF Heterodimer

Excision repair cross complementation group 1 (ERCC1) is an important protein in the nucleotide excision repair (NER) pathway, which is responsible for removing DNA adducts induced by platinum based compounds. The heterodimer ERCC1-XPF is one of two endonucleases required for NER. Genetic variations or polymorphisms in ERCC1 gene alter DNA repair capacity. Reduced DNA repair (NER) capacity may result in tumors and enhances cisplatin chemotherapy in cancer patients, which functions by causing DNA damage. Therefore, ERCC1 variants have the potential to be used as a strong candidate biomarker in cancer treatments. In this study we identified five variants V116M, R156Q, A199T, S267P, and R322C of ERCC1 gene as highly deleterious. Further structural and functional analysis has been conducted for ERCC1 protein in the presence of three variants V116M, R156Q, and A199T. Occurrence of theses variations adversely affected the regular interaction between ERCC1 and XPF protein. Analysis of 20 ns molecular dynamics simulation trajectories reveals that the predicted deleterious variants altered the ERCC1-XPF complex stability, flexibility, and surface area. Notably, the number of hydrogen bonds in ERCC1-XPF mutant complexes decreased in the molecular dynamic simulation periods. Overall, this study explores the link between the ERCC1 deleterious variants and cisplatin chemotherapy for various cancers with the help of molecular docking and molecular dynamic approaches.

Strand breakage of a (6–4) photoproduct-containing DNA at neutral pH and its repair by the ERCC1–XPF protein complex

The (6-4) photoproduct is one of the major UV-induced lesions in DNA. We previously showed that hydrolytic ring opening of the 5' base and subsequent hydrolysis of the glycosidic bond of the 3' component occurred when this photoproduct was treated with aqueous NaOH. In this study, we found that another product was obtained when the (6-4) photoproduct was heated at 90 °C for 6 h, in a 0.1 M solution of N,N'-dimethyl-1,2-ethanediamine adjusted to pH 7.4 with acetic acid. An analysis of the chemical structure of this product revealed that the 5' base was intact, whereas the glycosidic bond at the 3' component was hydrolyzed in the same manner. The strand break was detected for a 30-mer oligonucleotide containing the (6-4) photoproduct upon treatment with the above solution or other pH 7.4 solutions containing biogenic amines, such as spermidine and spermine. In the case of spermidine, the rate constant was calculated to be 1.4 × 10(-8) s(-1) at 37 °C. The strand break occurred even when the oligonucleotide was heated at 90 °C in 0.1 M sodium phosphate (pH 7.0), although this treatment produced several types of 5' fragments. The Dewar valence isomer was inert to this reaction. The product obtained from the (6-4) photoproduct-containing 30-mer was used to investigate the enzymatic processing of the 3' end bearing the damaged base and a phosphate. The ERCC1-XPF complex removed several nucleotides containing the damaged base, in the presence of replication protein A.

DNA repair endonuclease ERCC1-XPF as a novel therapeutic target to overcome chemoresistance in cancer therapy

The ERCC1–XPF complex is a structure-specific endonuclease essential for the repair of DNA damage by the nucleotide excision repair pathway. It is also involved in other key cellular processes, including DNA interstrand crosslink (ICL) repair and DNA double-strand break (DSB) repair. New evidence has recently emerged, increasing our understanding of its requirement in these additional roles. In this review, we focus on the protein–protein and protein–DNA interactions made by the ERCC1 and XPF proteins and discuss how these coordinate ERCC1–XPF in its various roles. In a number of different cancers, high expression of ERCC1 has been linked to a poor response to platinum-based chemotherapy. We discuss prospects for the development of DNA repair inhibitors that target the activity, stability or protein interactions of the ERCC1–XPF complex as a novel therapeutic strategy to overcome chemoresistance.

The ERCC1/XPF endonuclease is required for completion of homologous recombination at DNA replication forks stalled by inter-strand cross-links.

Both the ERCC1-XPF complex and the proteins involved in homoIogous recombination (HR) have critical roles in inter-strand cross-link (ICL) repair. Here, we report that mitomycin C-induced lesions inhibit replication fork elongation. Furthermore, mitomycin C-induced DNA double-strand breaks (DSBs) are the result of the collapse of ICL-stalled replication forks. These are not formed through replication run off, as we show that mitomycin C or cisplatin-induced DNA lesions are not incised by global genome nucleotide excision repair (GGR). We also suggest that ICL-lesion repair is initiated either by replication or transcription, as the GGR does not incise ICL-lesions. Furthermore, we report that RAD51 foci are induced by cisplatin or mitomycin C independently of ERCC1, but that mitomycin C-induced HR measured in a reporter construct is impaired in ERCC1-defective cells. These data suggest that ERCC1–XPF plays a role in completion of HR in ICL repair. We also find no additional sensitivity to cisplatin by siRNA co-depletion of XRCC3 and ERCC1, showing that the two proteins act on the same pathway to promote survival.

TERF2-XPF: Caught in the middle; beginnings from the end

Two recent articles suggest new roles for the TERF2-XPF complex (a.k.a. TRF2-XPF) 1 1 We take the liberty of referring to new evidence regarding the ERCC1-XPF complex, when the actual data has examined XPF only. in the recognition/repair of DNA damage at non-telomeric chromosomal locations (i.e. “Caught in the Middle”). These new roles for proteins typically ascribed functions at the ends of chromosomes are proposed to be very early events of DNA damage response (i.e. Beginnings from the End). Our previous understanding of a role for the TERF2-XPF complex in the maintenance of chromosome stability included the preservation of telomere length by “suppression” of the recognition of chromosome ends as breaks. One recent paper demonstrates that TERF2 also functions at non-telomeric sites of DNA damage, and does so prior to initiation of the ATM signaling cascade. A second paper goes on to demonstrate that overexpression of TERF2 produces mouse phenotypes similar to those associated with xeroderma pigmentosum, such as cellular hypersensitivity to UV radiation and DNA crosslinking agents, and telomere shortening and chromosome instability in response to DNA damage. Moreover, data are presented illustrating that these abnormal responses are not seen in an XPF −/− background, consistent with a dependency on XPF. Interestingly, both manuscripts focus on events that transpire in response to exogenous DNA damage. Here, we review these exciting findings that suggest new roles for the TERF2-XPF complex and point out several questions that remain to be addressed.

Functional and physical interactions between ERCC1 and MSH2 complexes for resistance to cis-diamminedichloroplatinum(II) in mammalian cells

Bulky DNA lesions are mainly repaired by nucleotide excision repair (NER), in which the interaction of ERCC1 with XPA protein recruits the ERCC1–XPF complex, which acts as a structure-specific endonuclease in the repair process. However, additional functions besides NER have been suggested for the ERCC1–XPF complex, because ERCC1- or XPF-deficient rodent cells are significantly more sensitive to DNA interstrand cross-linking (ICL) agents such as cis-diamminedichloroplatinum(II) (CDDP) than any other NER-deficient cells and because ERCC1-deficient mice suffer a more severe phenotype than XPA-deficient mice. By using RNA interference we show here that suppression of ERCC1 expression increases the sensitivity of xeroderma pigmentosum group A (XPA)-deficient human cells to CDDP but not to UV. This increased sensitivity to CDDP is observed in mouse cells defective in Xpa as well but not in cells defective both in Xpa and the mismatch repair gene Msh2. These data suggest that ERCC1 and MSH2 are involved co-operatively in CDDP resistance in mammalian cells. As a possible molecular basis, we show further a physical interaction between endogenous ERCC1 and MSH2 complexes in HeLa cell extracts. Using tagged ERCC1 in COS7 cells, the minimum region in ERCC1 necessary for the immuno-precipitation of MSH2 is turned out to be the carboxyl-terminal domain between the 184th and 260th amino acid, which is partly overlapping with the XPF-binding domain of ERCC1. This interaction may be important in additional functions of ERCC1–XPF including the repair of CDDP-induced DNA damage.

Conserved Residues of Human XPG Protein Important for Nuclease Activity and Function in Nucleotide Excision Repair

The human XPG endonuclease cuts on the 3' side of a DNA lesion during nucleotide excision repair. Mutations in XPG can lead to the disorders xeroderma pigmentosum (XP) and Cockayne syndrome. XPG shares sequence similarities in two regions with a family of structure-specific nucleases and exonucleases. To begin defining its catalytic mechanism, we changed highly conserved residues and determined the effects on the endonuclease activity of isolated XPG, its function in open complex formation and dual incision reconstituted with purified proteins, and its ability to restore cellular resistance to UV light. The substitution A792V present in two XP complementation group G (XP-G) individuals reduced but did not abolish endonuclease activity, explaining their mild clinical phenotype. Isolated XPG proteins with Asp-77 or Glu-791 substitutions did not cleave DNA. In the reconstituted repair system, alanine substitutions at these positions permitted open complex formation but were inactive for 3' cleavage, whereas D77E and E791D proteins retained considerable activity. The function of each mutant protein in the reconstituted system was mirrored by its ability to restore UV resistance to XP-G cell lines. Hydrodynamic measurements indicated that XPG exists as a monomer in high salt conditions, but immunoprecipitation of intact and truncated XPG proteins showed that XPG polypeptides can interact with each other, suggesting dimerization as an element of XPG function. The mutation results define critical residues in the catalytic center of XPG and strongly suggest that key features of the strand cleavage mechanism and active site structure are shared by members of the nuclease family.

DNA Structural Elements Required for ERCC1-XPF Endonuclease Activity

The heterodimeric complex ERCC1-XPF is a structure-specific endonuclease responsible for the 5' incision during mammalian nucleotide excision repair (NER). Additionally, ERCC1-XPF is thought to function in the repair of interstrand DNA cross-links and, by analogy to the homologous Rad1-Rad10 complex in Saccharomyces cerevisiae, in recombination between direct repeated DNA sequences. To gain insight into the role of ERCC1-XPF in such recombinational processes and in the NER reaction, we studied in detail the DNA structural elements required for ERCC1-XPF endonucleolytic activity. Recombinant ERCC1-XPF, purified from insect cells, was found to cleave stem-loop substrates at the DNA junction in the absence of other proteins like replication protein A, showing that the structure-specific endonuclease activity is intrinsic to the complex. Cleavage depended on the presence of divalent cations and was optimal in low Mn2+ concentrations (0.2 mM). A minimum of 4-8 unpaired nucleotides was required for incisions by ERCC1-XPF. Splayed arm and flap substrates were also cut by ERCC1-XPF, resulting in the removal of 3' protruding single-stranded arms. All incisions occurred in one strand of duplex DNA at the 5' side of a junction with single-stranded DNA. The exact cleavage position varied from 2 to 8 nucleotides away from the junction. One single-stranded arm, protruding either in the 3' or 5' direction, was necessary and sufficient for correct positioning of incisions by ERCC1-XPF. Our data specify the engagement of ERCC1-XPF in NER and allow a more direct search for its specific role in recombination.

Mechanism of open complex and dual incision formation by human nucleotide excision repair factors.

During nucleotide excision repair in human cells, a damaged DNA strand is cleaved by two endonucleases, XPG on the 3' side of the lesion and ERCC1-XPF on the 5' side. These structure-specific enzymes act at junctions between duplex and single-stranded DNA. ATP-dependent formation of an open DNA structure of approximately 25 nt around the adduct precedes this dual incision. We investigated the mechanism of open complex formation and find that mutations in XPB or XPD, the DNA helicase subunits of the transcription and repair factor TFIIH, can completely prevent opening and dual incision in cell-free extracts. A deficiency in XPC protein also prevents opening. The absence of RPA, XPA or XPG activities leads to an intermediate level of strand separation. In contrast, XPF or ERCC1-defective extracts open normally and generate a 3' incision, but fail to form the 5' incision. This same repair defect was observed in extracts from human xeroderma pigmentosum cells with an alteration in the C-terminal domain of XPB, suggesting that XPB has an additional role in facilitating 5' incision by ERCC1-XPF nuclease. These data support a mechanism in which TFIIH-associated helicase activity and XPC protein catalyze initial formation of the key open intermediate, with full extension to the cleavage sites promoted by the other core nucleotide excision repair factors. Opening is followed by dual incision, with the 3' cleavage made first.

Complementation of the DNA repair-deficient swi10 mutant of fission yeast by the human ERCC1 gene.

In human cells DNA damage caused by UV light is mainly repaired by the nucleotide excision repair pathway. This mechanism involves dual incisions on both sides of the damage catalyzed by two nucleases. In mammalian cells XPG cleaves 3' of the DNA lesion while the ERCC1-XPF complex makes the 5' incision. The amino acid sequence of the human excision repair protein ERCC1 is homologous with the fission yeast Swi10 protein. In order to test whether these proteins are functional homologues, we overexpressed the human gene in a Schizosaccharomyces pombe swi10 mutant. A swi10 mutation has a pleiotropic effect: it reduces the frequency of mating type switching (a mitotic transposition event from a silent cassette into the expression site) and causes increased UV sensitivity. We found that the full-length ERCC1 gene only complements the transposition defect of the fission yeast mutant, while a C-terminal truncated ERCC1 protein also restores the DNA repair capacity of the yeast cells. Using the two-hybrid system of Saccharomyces cerevisiae we show that only the truncated human ERCC1 protein is able to interact with the S . pombe Rad16 protein, which is the fission yeast homologue of human XPF. This is the first example yet known that a human gene can correct a yeast mutation in nucleotide excision repair.

The General Transcription-Repair Factor TFIIH Is Recruited to the Excision Repair Complex by the XPA Protein Independent of the TFIIE Transcription Factor

Recent studies have revealed that the general transcription factor TFIIH is also a general excision repair factor which, along with several other proteins, is required for transcription-independent excision reaction. As a general transcription factor, TFIIH is recruited to RNA polymerase II-promoter complex by another general transcription factor called TFIIE. We were interested in knowing whether TFIIE is also involved in recruiting TFIIH to the excision repair complex. We found that cell-free extract depleted of TFIIE carried out excision repair at a normal rate, leading us to conclude that TFIIE is not involved in recruiting TFIIH to the damage site and has no role in general excision repair. In contrast, the human damage recognition protein XPA specifically binds to TFIIH and apparently recruits it to the damage site. The carboxyl-terminal half of XPA is responsible for specific interaction with TFIIH. The C261S/C264S mutant of XPA bound the ERCC1-XPF complex normally, but failed to bind TFIIH and failed to complement an XP-A mutant cell-free extract indicating that the XPA-TFIIH interaction is essential to effecting the excision reaction. Interestingly, XPA also binds to the p34 subunit of TFIIE specifically and in competition with the p56 subunit of TFIIE. This latter interaction has no apparent role in general excision repair but may be relevant in the transcription-coupled repair reaction.