Cross-link Repair Research Articles

UVSSA facilitates transcription-coupled repair of DNA interstrand crosslinks

DNA interstrand crosslinks (ICLs) are covalent bonds between bases on opposing strands of the DNA helix which prevent DNA melting and subsequent DNA replication or RNA transcription. Here, we show that Ultraviolet Stimulated Scaffold Protein A (UVSSA) is critical for ICL repair in human cells, at least in part via the transcription coupled ICL repair (TC-ICR) pathway. Inactivation of UVSSA sensitizes human cells to ICL-inducing drugs, and delays ICL repair. UVSSA is required for replication-independent repair of a single ICL in a fluorescence-based reporter assay. UVSSA localizes to chromatin following ICL damage, and interacts with transcribing Pol II, CSA, CSB, and TFIIH. Specifically, UVSSA interaction with TFIIH is required for ICL repair and transcription inhibition blocks localization of transcription coupled repair factors to ICL damaged chromatin. Finally, UVSSA expression positively correlates with ICL-based chemotherapy resistance in human cancer cell lines. Our data strongly suggest that UVSSA is a novel ICL repair factor functioning in TC-ICR. These results provide further evidence that TC-ICR is a bona fide ICL repair mechanism that contributes to crosslinker drug resistance independently of replication-coupled ICL repair.

Variable clinical presentation of hypomorphic DCLRE1C deficiency from childhood to adulthood.

In this study, we aimed to report long-term follow-up of our pediatric and adult patients with DCLRE1C (DNA cross-link repair 1C) hypomorphic mutation who were diagnosed leaky severe combined immunodeficiency (SCID). Eighteen patients (13 children and five adults), aged between 6 and 29 years were included. Clinical and immunological features, including immunoglobulin levels, T and B cells, natural killer cell subsets, regulator T (Treg) cell ratios/markers, and cytokines, were assessed before and after hematopoietic stem cell transplantation (HSCT) and compared with healthy controls. Recurrent infections (78%) and skin manifestations (61%) such as granulomatous skin lesions, warts, and vitiligo were the most common clinical findings. Autoimmune diseases were observed in 33% and malignancy in 17%. Most patients had low serum IgA and B- and T-cell lymphopenia at the first admission. Recent thymic emigrants (RTE), Tnaive, Bnaive, CD56dimCD16+ cell ratios were significantly lower in the patients than in control; however, follicular helper T TFH and Th1 [interferon gamma (IFN-γ)] cell ratios were significantly higher than the control. Although, Treg ratio and its functional receptors tend to be high but not significant. Eleven patients (61.1%) were treated with HSCT. Median follow-up times of transplant patients was 56 (9-67) months. Patients with hypomorphic DCLRE1C mutations may present with variable clinical and laboratory findings at different ages. Our study showed a helper T (Th)1-dominant immune response before and after HSCT. Increased IFN-γ and TFH cells ratio could be a reason of chronic inflammation and autoimmunity developing before and after HSCT. Long-term follow-up of these patients after HSCT will help to better understand the disease and its pathophysiology.

Molecular insights into the stimulation of SNM1A nuclease activity by CSB during interstrand crosslink processing.

The SNM1A exonuclease plays a key role in repair of interstrand crosslinks (ICLs) which represent a particularly toxic class of DNA damage. Previous work suggests that the SWI/SNF family ATP-dependent, chromatin remodeler, Cockayne Syndrome B protein (CSB) interacts with SNM1A, during transcription-coupled DNA interstrand crosslink repair (TC-ICL repair). Here, we validate this interaction using purified proteins and demonstrate that the ubiquitin-binding and winged-helix domains of CSB are required for interaction with the catalytic domain of SNM1A. The winged helix domain is essential for binding, although high-affinity SNM1A binding requires the entire CSB C-terminal region (residues 1187-1493), where two copies of the C-terminal domain of CSB are necessary for a stable interaction with SNM1A. CSB stimulates SNM1A nuclease activity on varied model DNA repair intermediate substrates. Importantly, CSB was observed to stimulate digestion through ICLs in vitro , implying a key role of the interaction in 'unhooking' during TC-ICL repair. AlphaFold3 models of CSB constructs complexed with the SNM1A catalytic domain enabled mapping of the molecular contacts required for the CSB-SNM1A interaction. This identified specific protein-protein interactions necessary for CSB's stimulation of SNM1A's activity that we confirmed experimentally. Additionally, our studies reveal the C-terminal region of CSB as a novel DNA binding region that also is involved in stimulation of SNM1A-mediated ICL repair. Moreover, targeting protein-protein interactions that are vital for specific nuclease activities, such as CSB's stimulation of SNM1A's nuclease activity, may be a productive alternative therapeutic strategy to nuclease active site inhibition.

The Fanconi anemia core complex promotes CtIP-dependent end resection to drive homologous recombination at DNA double-strand breaks

During the repair of interstrand crosslinks (ICLs) a DNA double-strand break (DSB) is generated. The Fanconi anemia (FA) core complex, which is recruited to ICLs, promotes high-fidelity repair of this DSB by homologous recombination (HR). However, whether the FA core complex also promotes HR at ICL-independent DSBs, for example induced by ionizing irradiation or nucleases, remains controversial. Here, we identified the FA core complex members FANCL and Ube2T as HR-promoting factors in a CRISPR/Cas9-based screen. Using isogenic cell line models, we further demonstrated an HR-promoting function of FANCL and Ube2T, and of their ubiquitination substrate FANCD2. We show that FANCL and Ube2T localize at DSBs in a FANCM-dependent manner, and are required for the DSB accumulation of FANCD2. Mechanistically, we demonstrate that FANCL ubiquitin ligase activity is required for the accumulation of CtIP at DSBs, thereby promoting end resection and Rad51 loading. Together, these data demonstrate a dual genome maintenance function of the FA core complex and FANCD2 in promoting repair of both ICLs and DSBs.

Deciphering the role of post-translational modifications in fanconi anemia proteins and their influence on tumorigenesis.

Fanconi anemia (FA) is an autosomal or X-linked human disease, characterized by bone marrow failure, cancer susceptibility and various developmental abnormalities. So far, at least 22 FA genes (FANCA-W) have been identified. Germline inactivation of any one of these FA genes causes FA symptoms. Proteins encoded by FA genes are involved in the Fanconi anemia pathway, which is known for its roles in DNA inter-strand crosslinks (ICLs) repair. Besides, its roles in genome maintenance upon replication stress has also been reported. Post-translational modifications (PTMs) of FA proteins, particularly phosphorylation and ubiquitination, emerge as critical determinants in the activation of the FA pathway during ICL repair or replication stress response. Consequent inactivation of the FA pathway engenders heightened chromosomal instability, thereby constituting a genetic susceptibility conducive to cancer predisposition and the exacerbation of tumorigenesis. In this review, we have combined recent structural analysis of FA proteins and summarized knowledge on the functions of different PTMs in regulating FA pathways, and discuss potential contributions stemming from mutations at PTMs to the genesis and progression of tumorigenesis.

The role of SLFN11 in DNA replication stress response and its implications for the Fanconi anemia pathway

Fanconi anemia (FA) is a hereditary disorder characterized by a deficiency in the repair of DNA interstrand crosslinks and the response to replication stress. Endogenous DNA damage, most likely caused by aldehydes, severely affects hematopoietic stem cells in FA, resulting in progressive bone marrow failure and the development of leukemia. Recent studies revealed that expression levels of SLFN11 affect the replication stress response and are a strong determinant in cell killing by DNA-damaging cancer chemotherapy. Because SLFN11 is highly expressed in the hematopoietic system, we speculated that SLFN11 may have a significant role in FA pathophysiology. Indeed, we found that DNA damage sensitivity in FA cells is significantly mitigated by the loss of SLFN11 expression. Mechanistically, we demonstrated that SLFN11 destabilizes the nascent DNA strands upon replication fork stalling. In this review, we summarize our work regarding an interplay between SLFN11 and the FA pathway, and the role of SLFN11 in the response to replication stress.

The critical role of the iron-sulfur cluster and CTC components in DOG-1/BRIP1 function in Caenorhabditis elegans.

FANCJ/BRIP1, initially identified as DOG-1(Deletions Of G-rich DNA) in Caenorhabditiselegans, plays a critical role in genome integrity by facilitating DNA interstrand cross-link repair and resolving G-quadruplex structures. Its function is tightly linked to a conserved [4Fe-4S] cluster-binding motif, mutations of which contribute to Fanconi anemia and various cancers. This study investigates the critical role of the iron-sulfur (Fe-S) cluster in DOG-1 and its relationship with the cytosolic iron-sulfur protein assembly targeting complex (CTC). We found that a DOG-1 mutant, expected to be defective in Fe-S cluster binding, is primarily localized in the cytoplasm, leading to heightened DNA damage sensitivity and G-rich DNA deletions. We further discovered that the deletion of mms-19, a nonessential CTC component, also resulted in DOG-1 sequestered in cytoplasm and increased DNA damage sensitivity. Additionally, we identified that CIAO-1 and CIAO-2B are vital for DOG-1's stability and repair functions but unlike MMS-19 have essential roles in C. elegans. These findings confirm the CTC and Fe-S cluster as key elements in regulating DOG-1, crucial for genome integrity. Additionally, this study advances our understanding of the CTC's role in Fe-S protein regulation and development in C. elegans, offering a model to study its impact on multicellular organism development.

A Nutrigenomic View on the Premature-Aging Disease Fanconi Anemia.

Fanconi anemia, a rare disorder with an incidence of 1 in 300,000, is caused by mutations in FANC genes, which affect the repair of DNA interstrand crosslinks. The disease is characterized by congenital malformations, bone marrow failure within the first decade of life, and recurrent squamous cell carcinomas of the oral cavity, esophagus, and anogenital regions starting around age 20. In this review, we propose that Fanconi anemia should be considered a premature-aging syndrome. Interestingly, the onset and severity of the life-limiting clinical features of Fanconi anemia can be influenced by lifestyle choices, such as a healthy diet and physical activity. These factors shape the epigenetic status of at-risk cell types and enhance the competence of the immune system through nutritional signaling. Fanconi anemia may serve as a model for understanding the aging process in the general population, addressing research gaps in its clinical presentation and suggesting prevention strategies. Additionally, we will discuss how the balance of genetic and environmental risk factors-affecting both cancer onset and the speed of aging-is interlinked with signal transduction by dietary molecules. The underlying nutrigenomic principles will offer guidance for healthy aging in individuals with Fanconi anemia as well as for the general population.

Gemcitabine as chemotherapy of head and neck cancer in Fanconi anemia patients

Fanconi anemia (FA) is a rare hereditary disease resulting from an inactivating mutation in the FA/BRCA pathway, critical for the effective repair of DNA interstrand crosslinks (ICLs). The disease is characterized by congenital abnormalities, progressing bone marrow failure, and an increased risk of developing malignancies early in life, in particular head and neck squamous cell carcinoma (HNSCC). While ICL-inducing cisplatin combined with radiotherapy is a mainstay of HNSCC treatment, cisplatin is contra-indicated for FA-HNSCC patients. This dilemma necessitates the identification of novel treatment modalities tolerated by FA-HNSCC patients. To identify druggable targets, an siRNA-based genetic screen was previously performed in HNSCC-derived cell lines from FA and non-FA tumor origin. Here, we report that the Ribonucleotide Reductase (RNR) complex, consisting of the RRM1 and RRM2 subunits, was identified as a therapeutic target for both, FA and non-FA HNSCC. While non-FA HNSCC cells responded differentially to RNR depletion, FA-HNSCC cells were consistently found hypersensitive. This insight was confirmed pharmacologically using 2′, 2′-difluoro 2′deoxycytidine (dFdC), also known as gemcitabine, a clinically used nucleotide analog that is a potent inhibitor of the RNR complex. Importantly, while cisplatin exposure displayed severe, long-lasting toxicity on the hematopoietic stem and progenitor compartments in Fancg−/− mice, gemcitabine was well tolerated and had only a mild, transient impact. Taken together, our data implicate that gemcitabine-based chemoradiotherapy could serve as an alternative HNSCC treatment in Fanconi patients, and deserves clinical testing.

The interplay of homology-directed repair pathways in the repair of zebularine-induced DNA-protein crosslinks in Arabidopsis.

DNA-protein crosslinks (DPCs) are highly toxic DNA lesions represented by proteins covalently bound to the DNA. Persisting DPCs interfere with fundamental genetic processes such as DNA replication and transcription. Cytidine analog zebularine (ZEB) has been shown to crosslink DNA METHYLTRANSFERASE1 (MET1). Recently, we uncovered a critical role of the SMC5/6-mediated SUMOylation in the repair of DPCs. In an ongoing genetic screen, we identified two additional candidates, HYPERSENSITIVE TO ZEBULARINE 2 and 3, that were mapped to REGULATOR OF TELOMERE ELONGATION 1 (RTEL1) and polymerase TEBICHI (TEB), respectively. By monitoring the growth of hze2 and hze3 plants in response to zebularine, we show the importance of homologous recombination (HR) factor RTEL1 and microhomology-mediated end-joining (MMEJ) polymerase TEB in the repair of MET1-DPCs. Moreover, genetic interaction and sensitivity assays showed the interdependency of SMC5/6 complex, HR, and MMEJ in the homology-directed repair of MET1-DPCs in Arabidopsis. Altogether, we provide evidence that MET1-DPC repair in plants is more complex than originally expected.

Characterizing functional DNA damage and response caused by the combination of CHK1 and WEE1 inhibitors in ovarian and breast cancer models

BackgroundWe proposed to quantify reduction of functional DNA damage response (DDR) mechanisms caused by the combination of CHK1 and WEE1 inhibitors.MethodsSurvival of cells and tumor growth in-vitro and in-vivo caused by the combination of the CHK1 inhibitor SRA737 and the WEE1 inhibitor adavosertib was studied in OVCAR3 and MDA-MB 436 cells. Functional DNA damage was quantified using in vitro cell free DNA assays.ResultsThe combination of SRA737 and adavosertib caused significant reduction of survival of cells and DNA damage in-vitro and growth inhibition in-vivo. Studies using functional DDR assays found significant changes in the functional capacity of OVCAR3 but not MDA-MB 436 cells to repair DNA damage using multiple mechanisms including intra strand cross link repair, nucleotide excision repair, homologous recombination and non-homologous end joining. This study, for the first time provides a mechanistic insight into differences in the reduction in functional capacity of cells to repair DNA when exposed to CHK1 and WEE1 inhibitors.ConclusionThe combination of the CHK1 inhibitor SRA737 and WEE1 inhibitor adavosertib causes growth inhibition in-vitro and in-vivo, but differential functional inhibition of DDR in the models studied.

Abstract 7113: Targeting the repair of DNA-protein crosslinks induced by bifunctional alkylating agents

Abstract Bifunctional alkylating agents, such as nitrosoureas, platinum-based therapeutics, and chloroethylhydrazines, have demonstrated clinical successes against cancer by inducing cytotoxic interstrand or intrastrand DNA crosslinks. However, the less well-characterized capacity of these agents to couple DNA to chromosomally-associated proteins may also contribute to these compounds’ observed cytotoxicities. Bulky DNA-protein crosslinks (DPCs) could obstruct the DNA metabolism critical to cancer proliferation, thus contribute to genomic instability and cell death. To endogenously repair DPCs, human cells often utilize a DPC-specific proteolytic mechanism dependent on the metalloprotease SPRTN. Complementary approaches for quantifying DPCs, generated ex vivo, to assess the capacity of several bifunctional alkylating agents to induce these lesions in cultured human leukemia cells are presented here. In the first approach, proteins were isolated from lysates of drug-treated cells and protein-bound DNA was quantified as a ratio to the total DNA present in the lysates. In the complementary approach, DNA was extracted from lysates of drug-treated cells using a modified ‘STAR’ assay in which genomic DNA is isolated and the DNA-bound proteins were quantified by fluorescein isothiocyanate labeling. Micromolar concentrations of the nitrogen mustard mechlorethamine and nitrosourea lomustine induced significant DPC formation while more modest DPC formation was observed using similar concentrations of the chloroethylating agents carmustine and laromustine, as well as cisplatin. Bifunctional alkylating agents dependent on epoxide functionality, including dianhydrogalacticol and diepoxybutane, required drug concentrations of at least one order of magnitude larger to produce similar DPC yields measurable by these techniques. A strategy to interfere with the DPC repair pathway via SPRTN knockdown and assess increased sensitivity of cancer cells to bifunctional alkylating agents is also presented. Chemotherapeutic sensitization by targeting SPRTN could suggest a therapeutic strategy to improve effectiveness of several DNA-targeted anticancer agents, especially if extended to other therapeutics that trap DPCs such as inhibitors of topoisomerase and poly(ADP-ribose) polymerase. Further, identifying structure-function relationships with respect to bifunctional alkylating agents and DPC formation could inform the future development and clinical utilization of these chemotherapies. Citation Format: Cole Turner, Hailey Cerrato, Victoria Melehov, Charlotte Healy, Kevin P. Rice. Targeting the repair of DNA-protein crosslinks induced by bifunctional alkylating agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7113.

Mechanism of AGT-Mediated Repair of dG-dC Cross-Links in the Drug Resistance to Chloroethylnitrosoureas: Molecular Docking, MD Simulation, and ONIOM (QM/MM) Investigation.

Chloroethylnitrosoureas (CENUs) are important chemotherapies applied in the treatment of cancer. They exert anticancer activity by inducing DNA interstrand cross-links (ICLs) via the formation of two O6-alkylguanine intermediates, O6-chloroethylguanine (O6-ClEtG) and N1,O6-ethanoguanine (N1,O6-EtG). However, O6-alkylguanine-DNA alkyltransferase (AGT), a DNA-repair enzyme, can restore the O6-alkylguanine damages and thereby obstruct the formation of ICLs (dG-dC cross-link). In this study, the inhibitory mechanism of ICL formation was investigated to elucidate the drug resistance of CENUs mediated by AGT in detail. Based on the structures of the substrate-enzyme complexes obtained from docking and MD simulations, two ONIOM (QM/MM) models with different sizes of the QM region were constructed. The model with a larger QM region, which included the substrate (O6-ClEtG or N1,O6-EtG), a water molecule, and five residues (Tyr114, Cys145, His146, Lys165, and Glu172) in the active pocket of AGT, accurately described the repairing reaction and generated the results coinciding with the experimental outcomes. The repair process consists of two sequential steps: hydrogen transfer to form a thiolate anion on Cys145 and alkyl transfer from the O6 site of guanine (the rate-limiting step). The repair of N1,O6-EtG was more favorable than that of O6-ClEtG from both kinetics and thermodynamics aspects. Moreover, the comparison of the repairing process with the formation of dG-dC cross-link and the inhibition of AGT by O6-benzylguanine (O6-BG) showed that the presence of AGT could effectively interrupt the formation of ICLs leading to drug resistance, and the inhibition of AGT by O6-BG that was energetically more favorable than the repair of O6-ClEtG could not prevent the repair of N1,O6-EtG. Therefore, it is necessary to completely eliminate AGT activity before CENUs medication to enhance the chemotherapeutic effectiveness. This work provides reasonable explanations for the supposed mechanism of AGT-mediated drug resistance of CENUs and will assist in the development of novel CENU chemotherapies and their medication strategies.

Small Molecule Antagonists of the DNA Repair ERCC1/XPA Protein-Protein Interaction.

The DNA excision repair protein ERCC1 and the DNA damage sensor protein, XPA are highly overexpressed in patient samples of cisplatin-resistant solid tumors including lung, bladder, ovarian, and testicular cancer. The repair of cisplatin-DNA crosslinks is dependent upon nucleotide excision repair (NER) that is modulated by protein-protein binding interactions of ERCC1, the endonuclease, XPF, and XPA. Thus, inhibition of their function is a potential therapeutic strategy for the selective sensitization of tumors to DNA-damaging platinum-based cancer therapy. Here, we report on new small-molecule antagonists of the ERCC1/XPA protein-protein interaction (PPI) discovered using a high-throughput competitive fluorescence polarization binding assay. We discovered a unique structural class of thiopyridine-3-carbonitrile PPI antagonists that block a truncated XPA polypeptide from binding to ERCC1. Preliminary hit-to-lead studies from compound 1 reveal structure-activity relationships (SAR) and identify lead compound 27 o with an EC50 of 4.7 μM. Furthermore, chemical shift perturbation mapping by NMR confirms that 1 binds within the same site as the truncated XPA67-80 peptide. These novel ERCC1 antagonists are useful chemical biology tools for investigating DNA damage repair pathways and provide a good starting point for medicinal chemistry optimization as therapeutics for sensitizing tumors to DNA damaging agents and overcoming resistance to platinum-based chemotherapy.

Structure of Escherichia coli exonuclease VII

Exonuclease VII (ExoVII) is a ubiquitous bacterial nuclease. Encoded by the xseA and xseB genes, ExoVII participates in multiple nucleic acid-dependent pathways including the processing of multicopy single-stranded DNA and the repair of covalent DNA-protein crosslinks (DPCs). Although many biochemical properties of ExoVII have been defined, little is known about its structure/function relationships. Here, we use cryoelectron microscopy (cryoEM) to determine that Escherichia coli ExoVII comprises a highly elongated XseA4·XseB24 holo-complex. Each XseA subunit dimerizes through a central extended α-helical segment decorated by six XseB subunits and a C-terminal, domain-swapped β-barrel element; two XseA2·XseB12 subcomplexes further associate using N-terminal OB (oligonucleotide/oligosaccharide-binding) folds and catalytic domains to form a spindle-shaped, catenated octaicosamer. The catalytic domains of XseA, which adopt a nuclease fold related to 3-dehydroquinate dehydratases, are sequestered in the center of the complex and accessible only through large pores formed between XseA tetramers. The architectural organization of ExoVII, combined with biochemical studies, indicate that substrate selectivity is controlled by steric access to its nuclease elements and that tetramer dissociation results from substrate DNA binding. Despite a lack of sequence and fold homology, the physical organization of ExoVII is reminiscent of Mre11·Rad50/SbcCD ATP (adenosine triphosphate)-dependent nucleases used in the repair of double-stranded DNA breaks, including those formed by DPCs through aberrant topoisomerase activity, suggesting that there may have been convergent evolutionary pressure to contend with such damage events.

Novel Processes Associated with the Repair of Interstrand Cross-Links Derived from Abasic Sites in Duplex DNA: Roles for the Base Excision Repair Glycosylase NEIL3 and the SRAP Protein HMCES.

Recent studies have defined a novel pathway for the repair of interstrand cross-links derived from the reaction of an adenine residue with an apurinic/apyrimidinic (AP) site on the opposing strand of DNA (dA-AP ICL). Stalling of a replication fork at the dA-AP ICL triggers TRAIP-dependent ubiquitylation of the CMG helicase that recruits the base excision repair glycosylase NEIL3 to the lesion. NEIL3 unhooks the dA-AP ICL to regenerate the native adenine residue on one strand and an AP site on the other strand. Covalent capture of the abasic site by the SRAP protein HMCES protects against genomic instability that would result from cleavage of the abasic site in the context of single-stranded DNA at the replication fork. After repair synthesis moves the HMCES-AP adduct into the context of double-stranded DNA, the DNA-protein cross-link is resolved by a nonproteolytic mechanism involving dissociation of thiazolidine attachment. The AP site in duplex DNA is then repaired by the base excision repair pathway.

Purification and biochemical characterization of the G4 resolvase and DNA helicase FANCJ.

G-quadruplex (G4) DNA or RNA poses a unique nucleic acid structure in genomic transactions. Because of the unique topology presented by G4, cells have exquisite mechanisms and pathways to metabolize G4 that arise in guanine-rich regions of the genome such as telomeres, promoter regions, ribosomal DNA, and other chromosomal elements. G4 resolvases are often represented by a class of molecular motors known as helicases that disrupt the Hoogsteen hydrogen bonds in G4 by harnessing the chemical energy of nucleoside triphosphate hydrolysis. Of special interest to researchers in the field, including us, is the human FANCJ DNA helicase that efficiently resolves G4 DNA structures. Notably, FANCJ mutations are linked to Fanconi Anemia and are prominent in breast and ovarian cancer. Since our discovery that FANCJ efficiently resolves G4 DNA structures 15 years ago, we and other labs have characterized mechanistic aspects of FANCJ-catalyzed G4 resolution and its biological importance in genomic integrity and cellular DNA replication. In addition to its G4 resolvase function, FANCJ is also a classic DNA helicase that acts on conventional duplex DNA structures, which are relevant to the enzyme's role in interstrand cross link repair, double-strand break repair via homologous recombination, and response to replication stress. Here, we describe detailed procedures for the purification of recombinant FANCJ protein and characterization of its G4 resolvase and duplex DNA helicase activity.

The emergence of Fanconi anaemia type S: a phenotypic spectrum of biallelic BRCA1 mutations.

BRCA1 is involved in the Fanconi anaemia (FA) pathway, which coordinates repair of DNA interstrand cross-links. FA is a rare genetic disorder characterised by bone marrow failure, cancer predisposition and congenital abnormalities, caused by biallelic mutations affecting proteins in the FA pathway. Germline monoallelic pathogenic BRCA1 mutations are known to be associated with hereditary breast/ovarian cancer, however biallelic mutations of BRCA1 were long predicted to be incompatible with embryonic viability, hence BRCA1 was not considered to be a canonical FA gene. Despite this, several patients with biallelic pathogenic BRCA1 mutations and FA-like phenotypes have been identified - defining a new FA type (FA-S) and designating BRCA1 as an FA gene. This report presents a scoping review of the cases of biallelic BRCA1 mutations identified to date, discusses the functional effects of the mutations identified, and proposes a phenotypic spectrum of BRCA1 mutations based upon available clinical and genetic data. We report that this FA-S cohort phenotype includes short stature, microcephaly, facial dysmorphisms, hypo/hyperpigmented lesions, intellectual disability, chromosomal sensitivity to crosslinking agents and predisposition to breast/ovarian cancer and/or childhood cancers, with some patients exhibiting sensitivity to chemotherapy. Unlike most other types of FA, FA-S patients lack bone marrow failure.

Most Fanconi anemia heterozygotes are not at increased cancer risk: A genome-first DiscovEHR cohort population study

PurposeFanconi anemia (FA) is a bone marrow failure and cancer predisposition syndrome caused primarily by biallelic pathogenic variants in 1 of 22 genes involved in DNA interstrand cross-link repair. An enduring question concerns cancer risk of those with a single pathogenic FA gene variant. To investigate all FA genes, this study utilized the DiscovEHR cohort of 170,503 individuals with exome sequencing and electronic health data. Methods5822 subjects with a single pathogenic variant in an FA gene were identified. Two control groups were used in primary analysis deriving cancer risk signals. Secondary exploratory analysis was conducted using the UK Biobank and The Cancer Genome Atlas. ResultsSignals for elevated cancer risk were found in all 5 known cancer predisposition genes. Among the remaining 15 genes associated with autosomal recessive inheritance cancer risk signals were found for 4 cancers across 3 genes in the primary cohort but were not validated in secondary cohorts. ConclusionTo our knowledge, this is the first and largest FA heterozygote study to use genomic ascertainment and validates well-established cancer predispositions in 5 genes, whereas finding insufficient evidence of predisposition in 15 others. Our findings inform clinical surveillance given how common pathogenic FA variants are in the population.

Concepts of multi-level dynamical modelling: understanding mechanisms of squamous cell carcinoma development in Fanconi anemia.

Fanconi anemia (FA) is a rare disease (incidence of 1:300,000) primarily based on the inheritance of pathogenic variants in genes of the FA/BRCA (breast cancer) pathway. These variants ultimately reduce the functionality of different proteins involved in the repair of DNA interstrand crosslinks and DNA double-strand breaks. At birth, individuals with FA might present with typical malformations, particularly radial axis and renal malformations, as well as other physical abnormalities like skin pigmentation anomalies. During the first decade of life, FA mostly causes bone marrow failure due to reduced capacity and loss of the hematopoietic stem and progenitor cells. This often makes hematopoietic stem cell transplantation necessary, but this therapy increases the already intrinsic risk of developing squamous cell carcinoma (SCC) in early adult age. Due to the underlying genetic defect in FA, classical chemo-radiation-based treatment protocols cannot be applied. Therefore, detecting and treating the multi-step tumorigenesis process of SCC in an early stage, or even its progenitors, is the best option for prolonging the life of adult FA individuals. However, the small number of FA individuals makes classical evidence-based medicine approaches based on results from randomized clinical trials impossible. As an alternative, we introduce here the concept of multi-level dynamical modelling using large, longitudinally collected genome, proteome- and transcriptome-wide data sets from a small number of FA individuals. This mechanistic modelling approach is based on the "hallmarks of cancer in FA", which we derive from our unique database of the clinical history of over 750 FA individuals. Multi-omic data from healthy and diseased tissue samples of FA individuals are to be used for training constituent models of a multi-level tumorigenesis model, which will then be used to make experimentally testable predictions. In this way, mechanistic models facilitate not only a descriptive but also a functional understanding of SCC in FA. This approach will provide the basis for detecting signatures of SCCs at early stages and their precursors so they can be efficiently treated or even prevented, leading to a better prognosis and quality of life for the FA individual.