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.

Novel compound heterozygous variants in FANCI cause premature ovarian insufficiency.

Premature ovarian insufficiency (POI) is a common reproductive aging disorder due to a dramatic decline of ovarian function before 40 years of age. Accumulating evidence reveals that genetic defects, particularly those related to DNA damage response, are a crucial contributing factor to POI. We have demonstrated that the functional Fanconi anemia (FA) pathway maintains the rapid proliferation of primordial germ cells to establish a sufficient reproductive reserve by counteracting replication stress, but the clinical implications of this function in human ovarian function remain to be established. Here, we screened the FANCI gene, which encodes a key component for FA pathway activation, in our whole-exome sequencing database of 1030 patients with idiopathic POI, and identified two pairs of novel compound heterozygous variants, c.[97C > T];[1865C > T] and c.[158-2A > G];[c.959A > G], in two POI patients, respectively. The missense variants did not alter FANCI protein expression and nuclear localization, apart from the variant c.158-2A > G causing abnormal splicing and leading to a truncated mutant p.(S54Pfs*5). Furthermore, the four variants all diminished FANCD2 ubiquitination levels and increased DNA damage under replication stress, suggesting that the FANCI variants impaired FA pathway activation and replication stress response. This study first links replication stress response defects with the pathogenesis of human POI, providing a new insight into the essential roles of the FA genes in ovarian function.

FANCJ promotes PARP1 activity during DNA replication that is essential in BRCA1 deficient cells.

The effectiveness of poly (ADP-ribose) polymerase inhibitors (PARPi) in creating single-stranded DNA gaps and inducing sensitivity requires the FANCJ DNA helicase. Yet, how FANCJ relates to PARP1 inhibition or trapping, which contribute to PARPi toxicity, remains unclear. Here, we find PARPi effectiveness hinges on S-phase PARP1 activity, which is reduced in FANCJ deficient cells as G-quadruplexes sequester PARP1 and MSH2. Additionally, loss of the FANCJ-MLH1 interaction diminishes PARP1 activity; however, depleting MSH2 reinstates PARPi sensitivity and gaps. Indicating sequestered and trapped PARP1 are distinct, FANCJ loss increases PARPi resistance in cells susceptible to PARP1 trapping. However, with BRCA1 deficiency, the loss of FANCJ mirrors PARP1 loss or inhibition, with the detrimental commonality being loss of S-phase PARP1 activity. These insights underline the crucial role of PARP1 activity during DNA replication in BRCA1 deficient cells and emphasize the importance of understanding drug mechanisms for enhancing therapeutic response.

MutSß-MutLß-FANCJ axis mediates the restart of DNA replication after fork stalling at cotranscriptional G4/R-loops.

Transcription-replication conflicts (TRCs) induce formation of cotranscriptional RNA:DNA hybrids (R-loops) stabilized by G-quadruplexes (G4s) on the displaced DNA strand, which can cause fork stalling. Although it is known that these stalled forks can resume DNA synthesis in a process initiated by MUS81 endonuclease, how TRC-associated G4/R-loops are removed to allow fork passage remains unclear. Here, we identify the mismatch repair protein MutSß, an MLH1-PMS1 heterodimer termed MutLß, and the G4-resolving helicase FANCJ as factors that are required for MUS81-initiated restart of DNA replication at TRC sites in human cells. This DNA repair process depends on the G4-binding activity of MutSß, the helicase activity of FANCJ, and the binding of FANCJ to MLH1. Furthermore, we show that MutSß, MutLß, and MLH1-FANCJ interaction mediate FANCJ recruitment to G4s. These data suggest that MutSß, MutLß, and FANCJ act in conjunction to eliminate G4/R-loops at TRC sites, allowing replication restart.

Identification of a robust DNA methylation signature for Fanconi anemia.

Fanconi anemia (FA) is a clinically variable and genetically heterogeneous cancer-predisposing disorder representing the most common bone marrow failure syndrome. It is caused by inactivating predominantly biallelic mutations involving >20 genes encoding proteins with roles in the FA/BRCA DNA repair pathway. Molecular diagnosis of FA is challenging due to the wide spectrum of the contributing gene mutations and structural rearrangements. The assessment of chromosomal fragility after exposure to DNA cross-linking agents is generally required to definitively confirm diagnosis. We assessed peripheral blood genome-wide DNA methylation (DNAm) profiles in 25 subjects with molecularly confirmed clinical diagnosis of FA (FANCA complementation group) using Illumina's Infinium EPIC array. We identified 82 differentially methylated CpG sites that allow to distinguish subjects with FA from healthy individuals and subjects with other genetic disorders, defining an FA-specific DNAm signature. The episignature was validated using a second cohort of subjects with FA involving different complementation groups, documenting broader genetic sensitivity and demonstrating its specificity using the EpiSign Knowledge Database. The episignature properly classified DNA samples obtained from bone marrow aspirates, demonstrating robustness. Using the selected probes, we trained a machine-learning model able to classify EPIC DNAm profiles in molecularly unsolved cases. Finally, we show that the generated episignature includes CpG sites that do not undergo functional selective pressure, allowing diagnosis of FA in individuals with reverted phenotype due to gene conversion. These findings provide a tool to accelerate diagnostic testing in FA and broaden the clinical utility of DNAm profiling in the diagnostic setting.

Fanconi anemia associated protein 20 (FAAP20) plays an essential role in homology-directed repair of DNA double-strand breaks.

FAAP20 is a Fanconi anemia (FA) protein that associates with the FA core complex to promote FANCD2/FANCI monoubiquitination and activate the damage response to interstrand crosslink damage. Here, we report that FAAP20 has a marked role in homologous recombination at a DNA double-strand break not associated with an ICL and separable from its binding partner FANCA. While FAAP20's role in homologous recombination is not dependent on FANCA, we found that FAAP20 stimulates FANCA's biochemical activity in vitro and participates in the single-strand annealing pathway of double-strand break repair in a FANCA-dependent manner. This indicates that FAAP20 has roles in several homology-directed repair pathways. Like other homology-directed repair factors, FAAP20 loss causes a reduction in nuclear RAD51 Irradiation-induced foci; and sensitizes cancer cells to ionizing radiation and PARP inhibition. In summary, FAAP20 participates in DNA double strand break repair by supporting homologous recombination in a non-redundant manner to FANCA, and single-strand annealing repair via FANCA-mediated strand annealing activity.

Phosphorylation by ATR triggers FANCD2 chromatin loading and activates the Fanconi anemia pathway.

The Fanconi anemia (FA) pathway repairs DNA interstrand crosslinks (ICLs) in humans. Activation of the pathway relies on loading of the FANCD2/FANCI complex onto chromosomes, where it is fully activated by subsequent monoubiquitination. However, the mechanism for loading the complex onto chromosomes remains unclear. Here, we identify 10 SQ/TQ phosphorylation sites on FANCD2, which are phosphorylated by ATR in response to ICLs. Using a range of biochemical assays complemented with live-cell imaging including super-resolution single-molecule tracking, we show that these phosphorylation events are critical for loading of the complex onto chromosomes and for its subsequent monoubiquitination. We uncover how the phosphorylation events are tightly regulated in cells and that mimicking their constant phosphorylation leads to an uncontrolled active state of FANCD2, which is loaded onto chromosomes in an unrestrained fashion. Taken together, we describe a mechanism where ATR triggers FANCD2/FANCI loading onto chromosomes.

Spectrum of BRCA1 interacting helicase 1 aberrations and potential prognostic and therapeutic implication: a pan cancer analysis.

BRCA1 interacting helicase 1 (BRIP1) alteration was crucial in tumors and it was a potential therapeutic target in ovarian serous cystadenocarcinoma (OV). Although a small number of studies had focused on BRIP1, an extensive study of BRIP1 genetic mutation and its clinical application in different cancer types had not been analyzed. In the current study, we analyzed BRIP1 abnormal expression, methylation, mutation, and their clinical application via several extensive datasets, which covered over 10,000 tumor samples across more than 30 cancer types. The total mutation rate of BRIP1 was rare in pan cancer. Its alteration frequency, oncogenic effects, mutation, and therapeutic implications were different in each cancer. 242 BRIP1 mutations were found across 32 cancer types. UCEC had the highest alteration (mutation and CNV) frequency. In addition, BRIP1 was a crucial oncogenic factor in OV and BRCA. BRIP1 mutation in PRAD was targetable, and FDA had approved a new drug. Moreover, Kaplan-Meier curve analysis showed that BRIP1 expression and genetic aberrations were closely related to patient survival in several cancers, indicating their potential for application as new tumor markers and therapeutic targets. The current study profiled the total BRIP1 mutation spectrum and offered an extensive molecular outlook of BRIP1 in a pan cancer analysis. And it suggested a brand-new perspective for clinical cancer therapy.

UBE2T resolves transcription-replication conflicts and protects common fragile sites in primordial germ cells.

The proper development of primordial germ cells (PGCs) is an essential prerequisite for gametogenesis and mammalian fertility. The Fanconi anemia (FA) pathway functions in maintaining the development of PGCs. FANCT/UBE2T serves as an E2 ubiquitin-conjugating enzyme that ubiquitylates the FANCD2-FANCI complex to activate the FA pathway, but its role in the development of PGCs is not clear. In this study, we found that Ube2t knockout mice showed defects in PGC proliferation, leading to severe loss of germ cells after birth. Deletion of UBE2T exacerbated DNA damage and triggered the activation of the p53 pathway. We further demonstrated that UBE2T counteracted transcription-replication conflicts by resolving R-loops and stabilizing replication forks, and also protected common fragile sites by resolving R-loops in large genes and promoting mitotic DNA synthesis to maintain the genome stability of PGCs. Overall, these results provide new insights into the function and regulatory mechanisms of the FA pathway ensuring normal development of PGCs.

Multimolecular characteristics and role of BRCA1 interacting protein C-terminal helicase 1 (BRIP1) in human tumors: a pan-cancer analysis.

BACKGROUND: The aberrant expression of BRIP1 was associated with several cancers; however, the panoramic picture of BRIP1 in human tumors remains unclear. This study aims to explore the pan-cancerous picture of the expression of BRIP1 across 33 human cancers. METHODS: Based on the data from TCGA and GTEx, a series of bioinformatic analyses were applied to systematically explore the genetic landscape and biologic function of BRIP1 in 33 human tumors. RESULTS: We observed prognosis-related differential BRIP1 expressions between various carcinomas and the corresponding normal tissues. "Basal transcription factors," "homologous recombination," "nucleotide excision repair," and DNA metabolism pathways may play a role in the functional mechanisms of BRIP1. Patients with uterine corpus endometrial carcinoma presented with the highest alteration frequency of BRIP1 (nearly 10%). Single-nucleotide and copy number variations of BRIP1 were noticed in multiple cancers, and the expression of BRIP1 is significantly regulated by copy number variation in breast invasive carcinoma and lung squamous cell carcinoma. BRIP1 expression is negatively correlated with the DNA methylation levels in many tumors and is associated with the activation of apoptosis, cell cycle, DNA damage response, and inhibition of hormone ER and RNS/MARK signaling pathways. Moreover, a positive correlation was observed between BRIP1 expression and the immune infiltration levels of cancer-associated fibroblasts and CD8+ T cells in lung adenocarcinoma. CONCLUSION: Our pan-cancer analysis of BRIP1 provides a valuable resource for understanding the multimolecular characteristics and biological function of BRIP1 across human cancers.

Molecular Genetic Characteristics of FANCI, a Proposed New Ovarian Cancer Predisposing Gene.

FANCI was recently identified as a new candidate ovarian cancer (OC)-predisposing gene from the genetic analysis of carriers of FANCI c.1813C>T; p.L605F in OC families. Here, we aimed to investigate the molecular genetic characteristics of FANCI, as they have not been described in the context of cancer. We first investigated the germline genetic landscape of two sisters with OC from the discovery FANCI c.1813C>T; p.L605F family (F1528) to re-affirm the plausibility of this candidate. As we did not find other conclusive candidates, we then performed a candidate gene approach to identify other candidate variants in genes involved in the FANCI protein interactome in OC families negative for pathogenic variants in BRCA1, BRCA2, BRIP1, RAD51C, RAD51D, and FANCI, which identified four candidate variants. We then investigated FANCI in high-grade serous ovarian carcinoma (HGSC) from FANCI c.1813C>T carriers and found evidence of loss of the wild-type allele in tumour DNA from some of these cases. The somatic genetic landscape of OC tumours from FANCI c.1813C>T carriers was investigated for mutations in selected genes, copy number alterations, and mutational signatures, which determined that the profiles of tumours from carriers were characteristic of features exhibited by HGSC cases. As other OC-predisposing genes such as BRCA1 and BRCA2 are known to increase the risk of other cancers including breast cancer, we investigated the carrier frequency of germline FANCI c.1813C>T in various cancer types and found overall more carriers among cancer cases compared to cancer-free controls (p = 0.007). In these different tumour types, we also identified a spectrum of somatic variants in FANCI that were not restricted to any specific region within the gene. Collectively, these findings expand on the characteristics described for OC cases carrying FANCI c.1813C>T; p.L605F and suggest the possible involvement of FANCI in other cancer types at the germline and/or somatic level.

A DNA helicase remodeling proteins: How DNA-protein crosslink repair unfolds via FANCJ.

In this issue of Molecular Cell, Yaneva et al.1 demonstrate that the DNA helicase FANCJ promotes DNA replication-coupled DNA-protein crosslink (DPC) repair via an unexpected ability to unfold the protein adduct, thereby enabling its proteolysis by the DPC protease SPRTN.

Whole-exome sequencing identifies FANC heterozygous germline mutation as an adverse factor for immunosuppressive therapy in Chinese aplastic anemia patients aged 40 or younger: a single-center retrospective study.

Acquired aplastic anemia (AA) is a bone marrow failure disorder characterized by pancytopenia, and immunosuppressive therapy (IST) is the optional first-line management. Several studies identified the influencing factors on IST response; however, there are still a considerable number of patients suffering from poor prognoses. In this study, we enrolled 61 AA patients aged ≤ 40 years old, and whole-exome sequencing (WES) found unexpected high FANC heterozygous germline mutations (28/61, 45.9%). Patients with FANC mutations have a significantly lower absolute reticulocyte count and CD34+ % in the bone marrow and also lower 3-, 6-, and 9-month IST response than that without mutation, which were 0% vs. 25% (P = 0.017), 26.3% vs. 42.1% (P = 0.495), and 29.4% vs. 72.2% (P = 0.011), especially in anti-thymocyte globulin combined with the cyclosporin A (ATG + CsA) group, which were 0% vs.33.4% (P = 0.143), 25% vs.83.3% (P = 0.103), and 25% vs. 100% (P = 0.003), respectively. The event-free survival in the FANCwt group was also better than that in the FANCmut group (P = 0.016) and also showed in patients who received ATG + CsA treatment (P = 0.045). In addition, all the adverse effects of FANC germline mutation were not significant in stem cell-transplanted group. Our result indicated that the WES-based detection of FANC heterozygous germline mutations may have a great meaning in predicting IST response of acquired AA. This study was registered at chictr.org.cn (# ChiCTR2100054992).

The FANCC-FANCE-FANCF complex is evolutionarily conserved and regulates meiotic recombination.

At meiosis, programmed meiotic DNA double-strand breaks are repaired via homologous recombination, resulting in crossovers (COs). From a large excess of DNA double-strand breaks that are formed, only a small proportion gets converted into COs because of active mechanisms that restrict CO formation. The Fanconi anemia (FA) complex proteins AtFANCM, MHF1 and MHF2 were previously identified in a genetic screen as anti-CO factors that function during meiosis in Arabidopsis thaliana. Here, pursuing the same screen, we identify FANCC as a new anti-CO gene. FANCC was previously only identified in mammals because of low primary sequence conservation. We show that FANCC, and its physical interaction with FANCE-FANCF, is conserved from vertebrates to plants. Further, we show that FANCC, together with its subcomplex partners FANCE and FANCF, regulates meiotic recombination. Mutations of any of these three genes partially rescues CO-defective mutants, which is particularly marked in female meiosis. Functional loss of FANCC, FANCE, or FANCF results in synthetic meiotic catastrophe with the pro-CO factor MUS81. This work reveals that FANCC is conserved outside mammals and has an anti-CO role during meiosis together with FANCE and FANCF.

The Fanconi Anemia (FA) pathway is the subject of intense interest owing to the role of FA as a tumor suppressor. Three FA complex proteins, FANCM, MHF1 and MHF2, were identified as factors that suppress crossover during meiosis in the model plant Arabidopsis thaliana. Here, the authors extended these findings and identified a novel anti-crossover factor and showed that it encodes the plant FANCC homolog, which was previously thought to be vertebrate-specific. They further showed that FANCC regulates meiotic crossover together with two other FA proteins, FANCE and FANCF. This suggests that the FANCC­E­F subcomplex was already regulating DNA repair in the common ancestor of all living eukaryotes.

DNA repair protein FANCD2 has both ubiquitination-dependent and ubiquitination-independent functions during germ cell development.

When DNA interstrand crosslink lesions occur, a core complex of Fanconi anemia proteins promotes the ubiquitination of FANCD2 and FANCI, which recruit downstream factors to repair the lesion. However, FANCD2 maintains genome stability not only through its ubiquitination-dependent but also its ubiquitination-independent functions in various DNA damage response pathways. Increasing evidence suggests that FANCD2 is essential for fertility, but its ubiquitination-dependent and ubiquitination-independent roles during germ cell development are not well characterized. In this study, we analyzed germ cell development in Fancd2 KO and ubiquitination-deficient mutant (Fancd2K559R/K559R) mice. We showed that in the embryonic stage, both the ubiquitination-dependent and ubiquitination-independent functions of FANCD2 were required for the expansion of primordial germ cells and establishment of the reproductive reserve by reducing transcription-replication conflicts and thus maintaining genome stability in primordial germ cells. Furthermore, we found that during meiosis in spermatogenesis, FANCD2 promoted chromosome synapsis and regulated crossover formation independently of its ubiquitination, but that both ubiquitinated and nonubiquitinated FANCD2 functioned in programmed double strand break repair. Finally, we revealed that on meiotic XY chromosomes, H3K4me2 accumulation required ubiquitination-independent functionality of FANCD2, while the regulation of H3K9me2 and H3K9me3 depended on FANCD2 ubiquitination. Taken together, our findings suggest that FANCD2 has distinct functions that are both dependent on and independent of its ubiquitination during germ cell development.

Structural and biochemical basis of interdependent FANCI-FANCD2 ubiquitination.

Di-monoubiquitination of the FANCI-FANCD2 (ID2) complex is a central and crucial step for the repair of DNA interstrand crosslinks via the Fanconi anaemia pathway. While FANCD2 ubiquitination precedes FANCI ubiquitination, FANCD2 is also deubiquitinated at a faster rate than FANCI, which can result in a FANCI-ubiquitinated ID2 complex (IUb D2). Here, we present a 4.1 Å cryo-EM structure of IUb D2 complex bound to double-stranded DNA. We show that this complex, like ID2Ub and IUb D2Ub , is also in the closed ID2 conformation and clamps on DNA. The target lysine of FANCD2 (K561) becomes fully exposed in the IUb D2-DNA structure and is thus primed for ubiquitination. Similarly, FANCI's target lysine (K523) is also primed for ubiquitination in the ID2Ub -DNA complex. The IUb D2-DNA complex exhibits deubiquitination resistance, conferred by the presence of DNA and FANCD2. ID2Ub -DNA, on the other hand, can be efficiently deubiquitinated by USP1-UAF1, unless further ubiquitination on FANCI occurs. Therefore, FANCI ubiquitination effectively maintains FANCD2 ubiquitination in two ways: it prevents excessive FANCD2 deubiquitination within an IUb D2Ub -DNA complex, and it enables re-ubiquitination of FANCD2 within a transient, closed-on-DNA, IUb D2 complex.

Investigation of mutations in Fanconi anemia genes and malignancy predisposition in Brazilian patients.

INTRODUCTION: This study proposed to identify Fanconi anemia (FA) mutations in Brazilian patients and to investigate their impact on clinical manifestations and malignancies onset. METHODS: A total of 116 patients were screened for nine mutations in FANCA, FANCC, FANCG. Those with no mutations were investigated by multiplex ligation-dependent probe amplification (MLPA) and Sanger sequencing for FANCA, FANCC, FANCE, FANCF, FANCG, FANCD1/BRCA2. RESULTS: Genetic subtype was identified in 107/116 (78 FA-A, 8 FA-C, 13 FA-G, 8 FA-E), with only one mutation in 1/116, and no mutations in 9/116 patients. Before hematopoietic cell transplantation (HCT), malignancies were detected in 16/116 patients (14/78 FA-A, 01/08 FA-C, 01/08 FA-E), and 12 of them were hematological. Observed to expected ratio (O/E) of hematologic malignancy was 303.7 (95% CI = 148.6-458.7). CONCLUSION: This study allowed the identification of biallelic mutations in 91.4% of patients. FANCG and FANCC mutations had significantly earlier bone marrow failure onset, and FANCG severe cytopenia at diagnosis. Despite the inherent limitations of the small number of malignancy events in each genetic subtype, the hematologic malignancies O/E ratio was very high. Cumulative incidence of malignancy before HCT was higher in the third and fourth decades of life, considering HCT and death as competing risks. The cumulative incidence of HCT increased during the first decade, competing with malignancy development.

High expression levels of FANCI correlate with worse prognosis and promote tumor growth of lung adenocarcinoma partly via suppression of M1 macrophages.

FANCI, a member of the Fanconi anemia (FA) complementation group, normally associates with FANCD2 to play an important role in ribosome biogenesis and DNA repair. However, the correlation of FANCI with prognostic value and the molecular mechanism in patients with lung adenocarcinoma (LUAD) remains unclear. In the present study, bioinformatics analysis was performed on LUAD data from TCGA and GEO databases, and further confirmed by in vitro experiments. We found that a high level of FANCI was significantly correlated with a worse survival probability in patients with LUAD. Moreover, the results from in vitro experiments revealed high levels of FANCI in LUAD specimens and cell lines. Knockdown of FANCI expression in A549 and H460 cells significantly inhibited cell viability and clone formation of LUAD cells in vitro and in vivo. Furthermore, high FANCI levels were negatively correlated with a variety of tumor-infiltrating immune cells. Importantly, the overexpression of FANCI significantly inhibited the activation of M1 macrophages. All the data demonstrated that FANCI was a useful prognostic biomarker in patients with LUAD, and knockdown of FANCI inhibited tumor growth of LUAD cells in vitro and in vivo, partly by suppressing the activation of M1 macrophages.