ERCC1-XPF Endonuclease Research Articles

Exploring the Roles of Different DNA Repair Proteins in Short Inverted Repeat Mediated Genomic Instability: A Pilot Study

Repetitive DNA sequences are abundant in the human genome and can adopt alternative (i.e., non-B) DNA structures. These sequences contribute to diverse biological functions, including genomic instability. Previously, we found that Z-DNA-, H-DNA- and cruciform DNA-forming sequences are mutagenic, implicating them in cancer etiology. These sequences can stimulate the formation of DNA double-strand breaks (DSBs), causing deletions via cleavage by the endonuclease ERCC1-XPF. Interestingly, the activity of ERCC1-XPF in H-DNA-induced mutagenesis is nucleotide excision repair (NER)-dependent, but its role in Z-DNA-induced mutagenesis is NER-independent. Instead, Z-DNA is processed by ERCC1-XPF in a mechanism dependent on the mismatch repair (MMR) complex, MSH2-MSH3. These observations indicate distinct mechanisms of non-B-induced genomic instability. However, the roles of NER and MMR proteins, as well as additional nucleases (CtIP and MRE11), in the processing of cruciform DNA remain unknown. Here, we present data on the processing of cruciform-forming short inverted repeats (IRs) by DNA repair proteins using mammalian cell-based systems. From this pilot study, we show that, in contrast to H-DNA and Z-DNA, short IRs are processed in a NER- and MMR-independent manner, and the nucleases CtIP and MRE11 suppress short IR-induced genomic instability in mammalian cells.

Analysis of cytosine deamination events in excision repair sequencing reads reveals mechanisms of incision site selection in NER.

Nucleotide excision repair (NER) removes helix-distorting DNA lesions and is therefore critical for genome stability. During NER, DNA is unwound on either side of the lesion and excised, but the rules governing incision site selection, particularly in eukaryotic cells, are unclear. Excision repair-sequencing (XR-seq) sequences excised NER fragments, but analysis has been limited because the lesion location is unknown. Here, we exploit accelerated cytosine deamination rates in UV-induced CPD (cyclobutane pyrimidine dimer) lesions to precisely map their locations at C to T mismatches in XR-seq reads, revealing general and species-specific patterns of incision site selection during NER. Our data indicate that the 5' incision site occurs preferentially in HYV (i.e. not G; C/T;not T) sequence motifs, a pattern that can be explained by sequence preferences of the XPF-ERCC1 endonuclease. In contrast, the 3' incision site does not show strong sequence preferences, once truncated reads arising from mispriming events are excluded. Instead, the 3' incision is partially determined by the 5' incision site distance, indicating that the two incision events are coupled. Finally, our data reveal unique and coupled NER incision patterns at nucleosome boundaries. These findings reveal key principles governing NER incision site selection in eukaryotic cells.

A disease-associated XPA allele interferes with TFIIH binding and primarily affects transcription-coupled nucleotide excision repair

XPA is a central scaffold protein that coordinates the assembly of repair complexes in the global genome (GG-NER) and transcription-coupled nucleotide excision repair (TC-NER) subpathways. Inactivating mutations in XPA cause xeroderma pigmentosum (XP), which is characterized by extreme UV sensitivity and a highly elevated skin cancer risk. Here, we describe two Dutch siblings in their late forties carrying a homozygous H244R substitution in the C-terminus of XPA. They present with mild cutaneous manifestations of XP without skin cancer but suffer from marked neurological features, including cerebellar ataxia. We show that the mutant XPA protein has a severely weakened interaction with the transcription factor IIH (TFIIH) complex leading to an impaired association of the mutant XPA and the downstream endonuclease ERCC1-XPF with NER complexes. Despite these defects, the patient-derived fibroblasts and reconstituted knockout cells carrying the XPA-H244R substitution show intermediate UV sensitivity and considerable levels of residual GG-NER (~50%), in line with the intrinsic properties and activities of the purified protein. By contrast, XPA-H244R cells are exquisitely sensitive to transcription-blocking DNA damage, show no detectable recovery of transcription after UV irradiation, and display a severe deficiency in TC-NER-associated unscheduled DNA synthesis. Our characterization of a new case of XPA deficiency that interferes with TFIIH binding and primarily affects the transcription-coupled subpathway of nucleotide excision repair, provides an explanation of the dominant neurological features in these patients, and reveals a specific role for the C-terminus of XPA in TC-NER.

Modulation of ERCC1-XPF Heterodimerization Inhibition via Structural Modification of Small Molecule Inhibitor Side-Chains.

Inhibition of DNA repair enzymes is an attractive target for increasing the efficacy of DNA damaging chemotherapies. The ERCC1-XPF heterodimer is a key endonuclease in numerous single and double strand break repair processes, and inhibition of the heterodimerization has previously been shown to sensitize cancer cells to DNA damage. In this work, the previously reported ERCC1-XPF inhibitor 4 was used as the starting point for an in silico study of further modifications of the piperazine side-chain. A selection of the best scoring hits from the in silico screen were synthesized using a late stage functionalization strategy which should allow for further iterations of this class of inhibitors to be readily synthesized. Of the synthesized compounds, compound 6 performed the best in the in vitro fluorescence based endonuclease assay. The success of compound 6 in inhibiting ERCC1-XPF endonuclease activity in vitro translated well to cell-based assays investigating the inhibition of nucleotide excision repair and disruption of heterodimerization. Subsequently compound 6 was shown to sensitize HCT-116 cancer cells to treatment with UVC, cyclophosphamide, and ionizing radiation. This work serves as an important step towards the synergistic use of DNA repair inhibitors with chemotherapeutic drugs.

Mapping of the regions implicated in nuclear localization of multi-functional DNA repair endonuclease XPF-ERCC1.

The structure-specific endonuclease XPF-ERCC1 is a multi-functional heterodimer that participates in a variety of DNA repair mechanisms for maintaining genome integrity. Both subunits contain C-terminal tandem helix-hairpin-helix (HhH2 ) domains, which are necessary for not only their dimerization but also enzymatic activity as well as protein stability. However, the interdependency of both subunits in their nuclear localization remains poorly understood. In this study, we have analyzed the region(s) that affects the subcellular localization of XPF and ERCC1 using various deletion mutants. We first identified the nuclear localization signal (NLS) in XPF, which was essential for its nuclear localization under the ERCC1-free condition, but dispensable in the presence of ERCC1 (probably as XPF-ERCC1 heterodimer). Interestingly, in the NLS-independent and ERCC1-dependent XPF nuclear localization, the physical interaction between XPF and ERCC1 via C-terminal HhH2 domains was not needed. Instead, the amino acid regions 311-469 of XPF and 216-260 of ERCC1 are required for the nuclear localization. Furthermore, we found that the 311-469 region of XPF interacts with ERCC1 in a co-immunoprecipitation assay. These results suggest that the nuclear localization of XPF-ERCC1 heterodimer is regulated at multiple levels in an interdependent manner.

Research Highlights

C3 Complement Inhibition Prevents Antibody-mediated Rejection and Prolongs Renal Allograft Survival in Sensitized Nonhuman Primates Schmitz R, Fitch ZW, Schroder PM, et al. Nat Commun. 2021;12:5456. Current therapeutic strategies for desensitization in renal transplantation have shown limited efficacy.1 Because a major downstream effector arm of donor-specific antibody (DSA) is the complement cascade,2 complement inhibition strategies are gaining interest.3 Following C1 and C5 inhibition strategies, the C3 inhibitor Cp40 has recently been added to this field of promising candidates with a successful first-in-human study (NCT03316521) of its derivate AMY-101. In the study from Schmitz et al,4 Cp40 treatment was assessed in a nonhuman primate sensitized renal transplantation model, in which animals received donor skin transplants before the kidney transplant. Induction therapy with rhesus-specific antithymocyte globulin and maintenance therapy with tacrolimus, mycophenolate mofetil, and methylprednisolone were administered with (n = 6) or without (n = 5) peritransplant Cp40 treatment (day –2 to day 14). Although all animals showed a significant DSA increase after transplant, Cp40 treatment reduced acute antibody-mediated rejection and prolonged graft survival clearly (15.5 versus 4 d). Although initial lymphocyte depletion was not affected by Cp40, activation and proliferation proved to be markedly disrupted. Interestingly, virtually no intragraft C3d deposition could be detected during the treatment period with Cp40, which contrasted with controls and treated animals after discontinuation of Cp40. Within the treatment group, there were early (ie, during Cp40 treatment) and late rejectors. Although overall DSA levels were similar, late rejectors had less antibody-mediated rejection and less graft infiltration with CD68+ macrophages in parallel to downregulated complement-related genes. Late rejectors also had higher numbers of CD4+ CD25+ FoxP3+ regulatory T cells 1 wk after transplant, reduced T- and B-cell proliferation, and lower immunoglobulin (Ig)M DSAs with lower complement activation. The authors concluded that because of heterogeneity of IgM DSA levels, Cp40 doses could have been insufficient in animals bearing the highest immunologic burden. These data provide crucial evidence for C3 inhibition in a relevant preclinical model. Notably, overall DSA levels were not predictive of rejection in this study, yet treatment success correlated with IgM DSA load. This study sets the stage for early phase clinical trials of Cp40 in transplantation in which identification and stratification of potential benefit and dosage requirements in patient populations could be incorporated according to IgM DSA levels. An Aged Immune System Drives Senescence and Aging of Solid Organs Yousefzadeh MJ, Flores RR, Zhu Y, et al. Nature. 2021;594:100–105. The decline of immune responses with aging, termed immunosenescence, is linked to increased morbidity and mortality, likely related to reduced immunosurveillance, poor antipathogen immunity, and reduced vaccination responses.1,2 Given the broad role of the immune system in homeostasis, targeting immune cells to influence systemic aging may be a useful strategy for the reversal of age-related tissue pathology. In the landmark study from Yousefzadeh et al in Nature,3 the authors knocked out Ercc1 (which encodes the crucial DNA repair protein endonuclease ERCC1-XPF) in mouse hematopoietic cells, thereby selectively altering senescence in lymphoid organs. The impact of this genetic modification was evaluated in the context of aging. Compared with normal aging in wild-type mice, mutant mice developed peripheral leukopenia in adulthood and overall immune cells decreased in all compartments, as did thymic and splenic weights. Lymphoid tissues were less active and delayed-type hypersensitivity reactions demonstrated decreased innate and adaptive immune functions in older mutant mice, whereas younger ones had similar responses to wild-type littermates. Mutant mice displayed increased oxidative stress in lymphoid tissues and senescence onset in several immune cell subsets when compared with aged wild-type mice. Notably, older mutant mice had systemic changes associated with normal aging measured by DNA damage, oxidative stress, and senescence markers in many solid organ tissues, with peripheral senescence chronologically following immune senescence. Tissue damage appeared in nondeleted organs such as the liver, pancreas, intervertebral discs, brain, and muscles. Accordingly, the lifespan of mutant mice was markedly reduced. Interestingly, some of these effects could be transferred by simple transplantation of aged donor cells into separate recipient mice. Here, transplantation of aged or senescent splenocytes into reporter mice appeared to result in an acceleration of systemic aging and a decreased lifespan. Conversely, adoptive transfer of young wild-type splenocytes was able to attenuate peripheral organ senescence and tissue damage in older recipients. Finally, mechanistic target of rapamycin inhibition with rapamycin mitigated immune senescence characteristics connected with systemic aging. These findings underscore the driving role of immunosenescence in systemic aging, related tissue damage, and fatal outcomes. Additionally, the data highlight some of the molecular mechanisms linking aging and immunosenescence, which may be amenable to therapeutic targeting.

The 3'-flap endonuclease XPF-ERCC1 promotes alternative end joining and chromosomal translocation during B cell class switching.

Robust alternative end joining (A-EJ) in classical non-homologous end joining (c-NHEJ)-deficient murine cells features double-strand break (DSB) end resection and microhomology (MH) usage and promotes chromosomal translocation. The activities responsible for removing 3' single-strand overhangs following resection and MH annealing in A-EJ remain unclear. We show that, during class switch recombination (CSR) in mature mouse B cells, the structure-specific endonuclease complex XPF-ERCC1SLX4, although not required for normal CSR, represents a nucleotide-excision-repair-independent 3' flap removal activity for A-EJ-mediated CSR. B cells deficient in DNA ligase 4 and XPF-ERCC1 exhibit further impaired class switching, reducing joining to the resected S region DSBs without altering the MH pattern in S-S junctions. In ERCC1-deficient A-EJ cells, 3' single-stranded DNA (ssDNA) flaps that are generated predominantly in S/G2 phase of the cell cycle are susceptible to nuclease resolution. Moreover, ERCC1 promotes c-myc-IgH translocation in Lig4-/- cells. Our study reveals an important role of the flap endonuclease XPF-ERCC1 in A-EJ and oncogenic translocation in mouse B cells.

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.

XPF-ERCC1 protects liver, kidney and blood homeostasis outside the canonical excision repair pathways.

Loss of the XPF-ERCC1 endonuclease causes a dramatic phenotype that results in progeroid features associated with liver, kidney and bone marrow dysfunction. As this nuclease is involved in multiple DNA repair transactions, it is plausible that this severe phenotype results from the simultaneous inactivation of both branches of nucleotide excision repair (GG- and TC-NER) and Fanconi anaemia (FA) inter-strand crosslink (ICL) repair. Here we use genetics in human cells and mice to investigate the interaction between the canonical NER and ICL repair pathways and, subsequently, how their joint inactivation phenotypically overlaps with XPF-ERCC1 deficiency. We find that cells lacking TC-NER are sensitive to crosslinking agents and that there is a genetic interaction between NER and FA in the repair of certain endogenous crosslinking agents. However, joint inactivation of GG-NER, TC-NER and FA crosslink repair cannot account for the hypersensitivity of XPF-deficient cells to classical crosslinking agents nor is it sufficient to explain the extreme phenotype of Ercc1-/- mice. These analyses indicate that XPF-ERCC1 has important functions outside of its central role in NER and FA crosslink repair which are required to prevent endogenous DNA damage. Failure to resolve such damage leads to loss of tissue homeostasis in mice and humans.

Cryo-EM structures of the XPF-ERCC1 endonuclease reveal how DNA-junction engagement disrupts an auto-inhibited conformation

The structure-specific endonuclease XPF-ERCC1 participates in multiple DNA damage repair pathways including nucleotide excision repair (NER) and inter-strand crosslink repair (ICLR). How XPF-ERCC1 is catalytically activated by DNA junction substrates is not currently understood. Here we report cryo-electron microscopy structures of both DNA-free and DNA-bound human XPF-ERCC1. DNA-free XPF-ERCC1 adopts an auto-inhibited conformation in which the XPF helical domain masks the ERCC1 (HhH)2 domain and restricts access to the XPF catalytic site. DNA junction engagement releases the ERCC1 (HhH)2 domain to couple with the XPF-ERCC1 nuclease/nuclease-like domains. Structure-function data indicate xeroderma pigmentosum patient mutations frequently compromise the structural integrity of XPF-ERCC1. Fanconi anaemia patient mutations in XPF often display substantial in-vitro activity but are resistant to activation by ICLR recruitment factor SLX4. Our data provide insights into XPF-ERCC1 architecture and catalytic activation.

The XPF-ERCC1 Complex Is Essential for Genome Stability and Is Involved in the Mechanism of Gene Targeting in Physcomitrella patens.

The XPF-ERCC1 complex, a highly conserved structure-specific endonuclease, functions in multiple DNA repair pathways that are pivotal for maintaining genome stability, including nucleotide excision repair, interstrand crosslink repair, and homologous recombination. XPF-ERCC1 incises double-stranded DNA at double-strand/single-strand junctions, making it an ideal enzyme for processing DNA structures that contain partially unwound strands. Here, we have examined the role of the XPF-ERCC1 complex in the model bryophyte Physcomitrella patens which exhibits uniquely high gene targeting frequencies. We undertook targeted knockout of the Physcomitrella ERCC1 and XPF genes. Mutant analysis shows that the endonuclease complex is essential for resistance to UV-B and to the alkylating agent MMS, and contributes to the maintenance of genome integrity but is also involved in gene targeting in this model plant. Using different constructs we determine whether the function of the XPF-ERCC1 endonuclease complex in gene targeting was removal of 3′ non-homologous termini, similar to SSA, or processing of looped-out heteroduplex intermediates. Interestingly, our data suggest a role of the endonuclease in both pathways and have implications for the mechanism of targeted gene replacement in plants and its specificities compared to yeast and mammalian cells.

The role of SLX4 and its associated nucleases in DNA interstrand crosslink repair

A key step in the Fanconi anemia pathway of DNA interstrand crosslink (ICL) repair is the ICL unhooking by dual endonucleolytic incisions. SLX4/FANCP is a large scaffold protein that plays a central role in ICL unhooking. It contains multiple domains that interact with many proteins including three different endonucleases and also acts in several other DNA repair pathways. While it is known that its interaction with the endonuclease XPF-ERCC1 is required for its function in ICL repair, which other domains act in this process is unclear. Here, we used Xenopus egg extracts to determine ICL repair specific features of SLX4. We show that the SLX4-interacting endonuclease SLX1 is not required for ICL repair and demonstrate that all essential SLX4 domains are located at the N-terminal half of the protein. The MLR domain is crucial for the recruitment of XPF-ERCC1 but also has an unanticipated function in recruiting SLX4 to the site of damage. Although we find the BTB is not essential for ICL repair in our system, dimerization of SLX4 could be important. Our data provide new insights into the mechanism by which SLX4 acts in ICL repair.

Function and Interactions of ERCC1-XPF in DNA Damage Response.

Numerous proteins are involved in the multiple pathways of the DNA damage response network and play a key role to protect the genome from the wide variety of damages that can occur to DNA. An example of this is the structure-specific endonuclease ERCC1-XPF. This heterodimeric complex is in particular involved in nucleotide excision repair (NER), but also in double strand break repair and interstrand cross-link repair pathways. Here we review the function of ERCC1-XPF in various DNA repair pathways and discuss human disorders associated with ERCC1-XPF deficiency. We also overview our molecular and structural understanding of XPF-ERCC1.

Targeting the DNA Repair Endonuclease ERCC1-XPF with Green Tea Polyphenol Epigallocatechin-3-Gallate (EGCG) and Its Prodrug to Enhance Cisplatin Efficacy in Human Cancer Cells.

The 5′-3′ structure-specific endonuclease ERCC1/XPF (Excision Repair Cross-Complementation Group 1/Xeroderma Pigmentosum group F) plays critical roles in the repair of cisplatin-induced DNA damage. As such, it has been identified as a potential pharmacological target for enhancing clinical response to platinum-based chemotherapy. The goal of this study was to follow up on our previous identification of the compound NSC143099 as a potent inhibitor of ERCC1/XPF activity by performing an in silico screen to identify structural analogues that could inhibit ERCC1/XPF activity in vitro and in vivo. Using a fluorescence-based DNA-endonuclease incision assay, we identified the green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) as a potent inhibitor of ERCC1/XPF activity with an IC50 (half maximal inhibitory concentration) in the nanomolar range in biochemical assays. Using DNA repair assays and clonogenic survival assays, we show that EGCG can inhibit DNA repair and enhance cisplatin sensitivity in human cancer cells. Finally, we show that a prodrug of EGCG, Pro-EGCG (EGCG octaacetate), can enhance response to platinum-based chemotherapy in vivo. Together these data support a novel target of EGCG in cancer cells, namely ERCC1/XPF. Our studies also corroborate previous observations that EGCG enhances sensitivity to cisplatin in multiple cancer types. Thus, EGCG or its prodrug makes an ideal candidate for further pharmacological development with the goal of enhancing cisplatin response in human tumors.

Repair protein persistence at DNA lesions characterizes XPF defect with Cockayne syndrome features.

The structure-specific ERCC1-XPF endonuclease plays a key role in DNA damage excision by nucleotide excision repair (NER) and interstrand crosslink repair. Mutations in this complex can either cause xeroderma pigmentosum (XP) or XP combined with Cockayne syndrome (XPCS-complex) or Fanconi anemia. However, most patients carry compound heterozygous mutations, which confounds the dissection of the phenotypic consequences for each of the identified XPF alleles. Here, we analyzed the functional impact of individual pathogenic XPF alleles on NER. We show that XP-causing mutations diminish XPF recruitment to DNA damage and only mildly affect global genome NER. In contrast, an XPCS-complex-specific mutation causes persistent recruitment of XPF and the upstream core NER machinery to DNA damage and severely impairs both global genome and transcription-coupled NER. Remarkably, persistence of NER factors at DNA damage appears to be a common feature of XPCS-complex cells, suggesting that this could be a determining factor contributing to the development of additional developmental and/or neurodegenerative features in XP patients.

ERCC1-deficient cells and mice are hypersensitive to lipid peroxidation

Lipid peroxidation (LPO) products are relatively stable and abundant metabolites, which accumulate in tissues of mammals with aging, being able to modify all cellular nucleophiles, creating protein and DNA adducts including crosslinks. Here, we used cells and mice deficient in the ERCC1-XPF endonuclease required for nucleotide excision repair and the repair of DNA interstrand crosslinks to ask if specifically LPO-induced DNA damage contributes to loss of cell and tissue homeostasis. Ercc1−/− mouse embryonic fibroblasts were more sensitive than wild-type (WT) cells to the LPO products: 4-hydroxy-2-nonenal (HNE), crotonaldehyde and malondialdehyde. ERCC1-XPF hypomorphic mice were hypersensitive to CCl4 and a diet rich in polyunsaturated fatty acids, two potent inducers of endogenous LPO. To gain insight into the mechanism of how LPO influences DNA repair-deficient cells, we measured the impact of the major endogenous LPO product, HNE, on WT and Ercc1−/− cells. HNE inhibited proliferation, stimulated ROS and LPO formation, induced DNA base damage, strand breaks, error-prone translesion DNA synthesis and cellular senescence much more potently in Ercc1−/− cells than in DNA repair-competent control cells. HNE also deregulated base excision repair and energy production pathways. Our observations that ERCC1-deficient cells and mice are hypersensitive to LPO implicates LPO-induced DNA damage in contributing to cellular demise and tissue degeneration, notably even when the source of LPO is dietary polyunsaturated fats.

Spontaneous DNA damage to the nuclear genome promotes senescence, redox imbalance and aging

Accumulation of senescent cells over time contributes to aging and age-related diseases. However, what drives senescence in vivo is not clear. Here we used a genetic approach to determine if spontaneous nuclear DNA damage is sufficient to initiate senescence in mammals. Ercc1-/∆ mice with reduced expression of ERCC1-XPF endonuclease have impaired capacity to repair the nuclear genome. Ercc1-/∆ mice accumulated spontaneous, oxidative DNA damage more rapidly than wild-type (WT) mice. As a consequence, senescent cells accumulated more rapidly in Ercc1-/∆ mice compared to repair-competent animals. However, the levels of DNA damage and senescent cells in Ercc1-/∆ mice never exceeded that observed in old WT mice. Surprisingly, levels of reactive oxygen species (ROS) were increased in tissues of Ercc1-/∆ mice to an extent identical to naturally-aged WT mice. Increased enzymatic production of ROS and decreased antioxidants contributed to the elevation in oxidative stress in both Ercc1-/∆ and aged WT mice. Chronic treatment of Ercc1-/∆ mice with the mitochondrial-targeted radical scavenger XJB-5–131 attenuated oxidative DNA damage, senescence and age-related pathology. Our findings indicate that nuclear genotoxic stress arises, at least in part, due to mitochondrial-derived ROS, and this spontaneous DNA damage is sufficient to drive increased levels of ROS, cellular senescence, and the consequent age-related physiological decline.

Disruption of DNA repair in cancer cells by ubiquitination of a destabilising dimerization domain of nucleotide excision repair protein ERCC1.

DNA repair pathways present in all cells serve to preserve genome stability, but in cancer cells they also act reduce the efficacy of chemotherapy. The endonuclease ERCC1-XPF has an important role in the repair of DNA damage caused by a variety of chemotherapeutic agents and there has been intense interest in the use of ERCC1 as a predictive marker of therapeutic response in non-small cell lung carcinoma, squamous cell carcinoma and ovarian cancer. We have previously validated ERCC1 as a therapeutic target in melanoma, but all small molecule ERCC1-XPF inhibitors reported to date have lacked sufficient potency and specificity for clinical use. In an alternative approach to prevent the repair activity of ERCC1-XPF, we investigated the mechanism of ERCC1 ubiquitination and found that the key region was the C-terminal (HhH)2 domain which heterodimerizes with XPF. This ERCC1 region was modified by non-conventional lysine-independent, but proteasome-dependent polyubiquitination, involving Lys33 of ubiquitin and a linear ubiquitin chain. XPF was not polyubiquitinated and its expression was dependent on presence of ERCC1, but not vice versa. To our surprise we found that ERCC1 can also homodimerize through its C-terminal (HhH)2 domain. We exploited the ability of a peptide containing this C-terminal domain to destabilise both endogenous ERCC1 and XPF in human melanoma cells and fibroblasts, resulting in reductions of up to 85% in nucleotide excision repair and near two-fold increased sensitivity to DNA damaging agents. We suggest that the ERCC1 (HhH)2 domain could be used in an alternative strategy to treat cancer.

Abstract LB-265: 5’ gap-mediated long patch base excision repair

Abstract The base excision repair (BER) pathway is one of the most frequently used DNA repair mechanisms in the cell and modulates many human pathophysiological conditions related to DNA damage. Through live cell and in vitro reconstitution experiments, we have discovered a novel step in a sub-pathway of the conventional long-patch BER that involves formation of a 9 nt gap 5’ to the lesion, which is mediated by RECQ1 DNA helicase and ERCC1-XPF endonuclease in cooperation with PARP1 and RPA. The novel step is employed during repair of a variety of DNA lesions, including oxidative and alkylation damage. Moreover, RECQ1 regulates the autoribosylation status of PARP1 and the choice between long-patch and single-nucleotide BER, which regulates cellular toxicity to DNA damage. Taken together, our results propose a revised model of the long-patch BER pathway and characterize a crucial regulation point for pathway choice in BER. Citation Format: Jordan Woodrick, Sharon Camacho, Swetha Parvathaneni, Sudha Sharma, Rabindra Roy. 5’ gap-mediated long patch base excision repair [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-265. doi:10.1158/1538-7445.AM2017-LB-265

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.