Repair Of Interstrand Crosslinks Research Articles

FANCI plays an essential role in spermatogenesis and regulates meiotic histone methylation

FANCI is an essential component of Fanconi anemia pathway, which is responsible for the repair of DNA interstrand cross-links (ICLs). As an evolutionarily related partner of FANCD2, FANCI functions together with FANCD2 downstream of FA core complex. Currently, growing evidences showed that the essential role of FA pathway in male fertility. However, the underlying mechanisms for FANCI in regulating spermatogenesis remain unclear. In the present study, we found that the male Fanci−/− mice were sterile and exhibited abnormal spermatogenesis, including massive germ cell apoptosis in seminiferous tubules and dramatically decreased number of sperms in epididymis. Besides, FANCI deletion impaired maintenance of undifferentiated spermatogonia. Further investigation indicated that FANCI was essential for FANCD2 foci formation and regulated H3K4 and H3K9 methylation on meiotic sex chromosomes. These findings elucidate the role and mechanism of FANCI during spermatogenesis in mice and provide new insights into the etiology and molecular basis of nonobstructive azoospermia.

Histone demethylase AMX-1 is necessary for proper sensitivity to interstrand crosslink DNA damage.

Histone methylation is dynamically regulated to shape the epigenome and adjust central nuclear processes including transcription, cell cycle control and DNA repair. Lysine-specific histone demethylase 2 (LSD2) has been implicated in multiple types of human cancers. However, its functions remain poorly understood. This study investigated the histone demethylase LSD2 homolog AMX-1 in C. elegans and uncovered a potential link between H3K4me2 modulation and DNA interstrand crosslink (ICL) repair. AMX-1 is a histone demethylase and mainly localizes to embryonic cells, the mitotic gut and sheath cells. Lack of AMX-1 expression resulted in embryonic lethality, a decreased brood size and disorganized premeiotic tip germline nuclei. Expression of AMX-1 and of the histone H3K4 demethylase SPR-5 is reciprocally up-regulated upon lack of each other and the mutants show increased H3K4me2 levels in the germline, indicating that AMX-1 and SPR-5 regulate H3K4me2 demethylation. Loss of AMX-1 function activates the CHK-1 kinase acting downstream of ATR and leads to the accumulation of RAD-51 foci and increased DNA damage-dependent apoptosis in the germline. AMX-1 is required for the proper expression of mismatch repair component MutL/MLH-1 and sensitivity against ICLs. Interestingly, formation of ICLs lead to ubiquitination-dependent subcellular relocalization of AMX-1. Taken together, our data suggest that AMX-1 functions in ICL repair in the germline.

PFKFB3 Inhibition Sensitizes DNA Crosslinking Chemotherapies by Suppressing Fanconi Anemia Repair.

Simple SummaryDNA-damaging chemotherapeutics, such as platinum drugs, are cornerstones in cancer treatment. The efficacy of such treatment is intimately linked to the DNA repair capacity of the cancer cells, as DNA damage above a tolerable threshold culminates in cell death. Cancer cells often have deregulated DNA repair mechanisms, making them initially more sensitive to DNA-damaging chemotherapies. Unfortunately, over time, cancer cells often develop resistance to such treatments by rewiring their DNA damage response pathways. Here, we identify that targeting the recognized anti-cancer target 6-phosphofructo-2-kinase/fructose-2,6,-bisphophatase 3 (PFKFB3), commonly overexpressed in cancer, with the small molecule inhibitor KAN0438757, selectively sensitizes cancer cells to platinum drugs, including treatment-resistant cancer cells, while sparing normal cells. Mechanistically, PFKFB3 promotes tolerance to and the repair of platinum-induced DNA interstrand crosslinks (ICLs) through modulation of the Fanconi anemia (FA) DNA repair pathway. Thus targeting PFKFB3 opens up therapeutic possibilities to improve the efficacy of ICL-inducing cancer treatments.Replicative repair of interstrand crosslinks (ICL) generated by platinum chemotherapeutics is orchestrated by the Fanconi anemia (FA) repair pathway to ensure resolution of stalled replication forks and the maintenance of genomic integrity. Here, we identify novel regulation of FA repair by the cancer-associated glycolytic enzyme PFKFB3 that has functional consequences for replication-associated ICL repair and cancer cell survival. Inhibition of PFKFB3 displays a cancer-specific synergy with platinum compounds in blocking cell viability and restores sensitivity in treatment-resistant models. Notably, the synergies are associated with DNA-damage-induced chromatin association of PFKFB3 upon cancer transformation, which further increases upon platinum resistance. FA pathway activation triggers the PFKFB3 assembly into nuclear foci in an ATR- and FANCM-dependent manner. Blocking PFKFB3 activity disrupts the assembly of key FA repair factors and consequently prevents fork restart. This results in an incapacity to replicate cells to progress through S-phase, an accumulation of DNA damage in replicating cells, and fork collapse. We further validate PFKFB3-dependent regulation of FA repair in ex vivo cultures from cancer patients. Collectively, targeting PFKFB3 opens up therapeutic possibilities to improve the efficacy of ICL-inducing cancer treatments.

Mechanism, specificity, and function of FANCD2-FANCI ubiquitination and deubiquitination.

Fanconi anemia (FA) is a rare genetic disorder caused by mutations in any of the currently 22 known FA genes. The products of these genes, along with other FA-associated proteins, participate in a biochemical pathway, known as the FA pathway. This pathway is responsible for the repair of DNA interstrand cross-links (ICL) and the maintenance of genomic stability in response to replication stress. At the center of the pathway is the monoubiquitination of two FA proteins, FANCD2 and FANCI, on two specific lysine residues. This is achieved by the combined action of the UBE2T ubiquitin-conjugating enzyme and a large multicomponent E3 ligase, known as the FA-core complex. This E2-E3 pair specifically targets the FANCI-FANCD2 heterodimer (ID2 complex) for ubiquitination on DNA. Deubiquitination of both FANCD2 and FANCI, which is also critical for ICL repair, is achieved by the USP1-UAF1 complex. Recent work suggests that FANCD2 ubiquitination transforms the ID2 complex into a sliding DNA clamp. Further ubiquitination on FANCI does not alter this closed-on-DNA ID2 conformation. However, the resulting dimonoubiquitinated complex is highly resistant to USP1-UAF1 deubiquitination. This review will provide an update on recent work focusing on how specificity in FANCD2 ubiquitination and deubiquitination is achieved. Recent findings shedding light to the mechanisms, molecular functions, and biological roles of FANCI/FANCD2 ubiquitination and deubiquitination will be also discussed. ENZYMES: UBA1 (6.2.1.45), UBE2T (2.3.2.23), FANCL (2.3.2.27), USP1 (3.4.19.12).

MCM2-7 and FANCD2 Are Required for RAD51 Function in Homologous Recombination and ICL Repair

MCM2-7 helicase is required for DNA replication and has been implicated in the maintenance of genomic instability, as more than 90% of MCM complexes are in dormant origins. Decreased levels of MCM2-7 complex on chromatin lead to genomic instability. The Fanconi anemia (FA) pathway, whose proteins are defective in the genetic disorder that entails bone marrow failure, congenital defects, and acute myeloid leukemia, is also important in maintaining genomic instability through the regulation of homologous recombinatorial repair (HRR) of interstrand cross links (ICL). FANCD2 is the central protein in the FA pathway, exhibiting monoubiquitination in response to DNA damage, and has been shown to interact with MCM2-7. Here we show that monoubiquitinated FANCD2 interacts with MCM2-7 and is required for maintenance of MCM2-7 on chromatin upon DNA interstrand crosslinking (ICL) drug mitomycin C treatment. FANCD2-MCM2-7 also interacts with RAD51, an interaction which is decreased in the absence of FANCD2 or with the expression of the ubiquitination mutant FANCD2(K561R). Also, RAD51 chromatin localization and homologous recombination (HR) function is impaired in MCM7 knockdown cells. MCM2, MCM5, or MCM7 knock down cells exhibit hypersensitive to ICL and G2/M accumulation as well, and the hypersensitivity of MCM7 knock down cells can be rescued by re-expressing MCM7. Importantly FANCD2 and MCM7 are epistatic for MMC hypersensitivity. At the levels of MCM7 knock down, we observe that cells are not defective for replication as measured by BrdU labeling and are not hypersensitive to replication inhibitor hydroxyurea (HU) but remain ICL sensitive. Using recombinant protein reconstitution in vitro, we observe MCM2-7 and FANCI-FANCD2 dependent RAD51 filament stabilization and strand exchange, which are key steps in HRR. MCM2-7 proteins defective for helicase activity and thus replication defective remain functional in these in vitro biochemical assays. On the other hand, a FANCI DNA binding mutant completely abrogates these activities, suggesting that the FANCD2-FANCI-MCM2-7-RAD51 complex requires a regulated interaction with DNA and is separable from MCM2-7 replication function. Taken together, the FA pathway, MCM2-7, and RAD51 work cooperatively in the biochemical and cell response to DNA damage ICL. DisclosuresNo relevant conflicts of interest to declare.

Lnk/Sh2b3 Deficiency Restores Hematopoietic Stem Cell Function and Genome Integrity in Fanconi Anemia

▪Although initially identified over 85 years ago, Fanconi Anemia (FA) remains a fatal genetic disease. Patients with FA exhibit developmental malformations, bone marrow failure, and increased cancer susceptibility. Twenty-one FA genes cooperate in a genome stability pathway that is essential for repair of DNA interstrand crosslinks (ICLs) and attenuation of replication stress. Cells derived from FA patients are hypersensitive to ICL-inducing agents such as Mitomycin C (MMC), a hallmark of FA. In addition, FA hematopoietic stem and progenitor cells (HSPCs) show growth retardation and increased apoptosis culminating in hematopoietic stem cell (HSC) exhaustion and bone marrow failure. The poor growth of HSPCs also poses a significant obstacle in gene therapy for FA patients. Moreover, unrepaired DNA damage increases genome instability and promotes leukemia and cancer. Of note, interventions to mitigate HSPC defects in FA do not exist, aside from allogeneic bone marrow transplantation.Remarkably, we reveal here that loss of the adaptor protein Lnk (also called Sh2b3) suppresses FA associated phenotypes without further accelerating neoplastic transformation. LNK is a negative regulator of HSC self-renewal in part by attenuating cytokine signaling, in particular thrombopoietin (TPO)-activated JAK2 signaling in HSCs. Fancd2-/-;Lnk-/- mice exhibit increased phenotypic HSCs in comparison to wildtype (WT) animals. The reconstituting ability of Fancd2-/-;Lnk-/- HSCs is also largely restored to WT levels in serial transplantation assays. In addition, Lnk deficiency restored the growth and survival of FA HSPCs in ex vivo culture. More importantly, Lnk deficiency mitigated chromosomal aberrations associated with FA upon proliferation. To determine if Lnk deficiency mitigates ICL repair, we examined MMC sensitivity in primary HSPCs that were dually null for Lnk and FancD2. Our data suggest that LNK does not play an overt role in ICL repair. Rather, Lnk deficiency in HSPCs mitigated replication stress by stabilizing replication forks and reduces genome instability associated with FA. Hence, our data suggest that Lnk deficiency alleviates replication stress-induced DNA damage and genome instability, thereby restoring cell proliferation and survival of Fancd2-/- HSPCs ex vivo and HSC functions in vivo.Last and importantly, we began to test if LNK inhibition restores human FA HSPCs. We first generated FA-like HSPCs by knocking down FANCD2 in normal umbilical cord blood (UCB) CD34+ cells. Our results demonstrated that shRNA-mediated depletion of LNK in human CD34+ cells rescued progenitor cell numbers and growth in FA-like HSPCs. Take together, our findings reveal a previously unappreciated role of cytokine/JAK signaling in alleviating replication stress and promoting replication-associated genome maintenance. Our studies also illuminate replication stress as a major underlying origin of bone marrow failure in FA patients and have implications for therapeutic strategies to treat FA associated bone marrow failure. DisclosuresNo relevant conflicts of interest to declare.

PrimPol‐mediated repriming facilitates replication traverse of DNA interstrand crosslinks

DNA interstrand crosslinks (ICLs) induced by endogenous aldehydes or chemotherapeutic agents interfere with essential processes such as replication and transcription. ICL recognition and repair by the Fanconi Anemia pathway require the formation of an X‐shaped DNA structure that may arise from convergence of two replication forks at the crosslink or traversing of the lesion by a single replication fork. Here, we report that ICL traverse strictly requires DNA repriming events downstream of the lesion, which are carried out by PrimPol, the second primase‐polymerase identified in mammalian cells after Polα/Primase. The recruitment of PrimPol to the vicinity of ICLs depends on its interaction with RPA, but not on FANCM translocase or the BLM/TOP3A/RMI1‐2 (BTR) complex that also participate in ICL traverse. Genetic ablation of PRIMPOL makes cells more dependent on the fork convergence mechanism to initiate ICL repair, and PRIMPOL KO cells and mice display hypersensitivity to ICL‐inducing drugs. These results open the possibility of targeting PrimPol activity to enhance the efficacy of chemotherapy based on DNA crosslinking agents.

Fanconi anemia proteins participate in a break-induced-replication-like pathway to counter replication stress.

Fanconi anemia (FA) is a devastating hereditary disease characterized by bone marrow failure (BMF) and acute myeloid leukemia (AML). As FA-deficient cells are hypersensitive to DNA interstrand crosslinks (ICLs), ICLs are widely assumed to be the lesions responsible for FA symptoms. Here, we show that FA-mutated cells are hypersensitive to persistent replication stress and that FA proteins play a role in the break-induced-replication (BIR)-like pathway for fork restart. Both the BIR-like pathway and ICL repair share almost identical molecular mechanisms of 53BP1-BRCA1-controlled signaling response, SLX4- and FAN1-mediated fork cleavage and POLD3-dependent DNA synthesis, suggesting that the FA pathway is intrinsically one of the BIR-like pathways. Replication stress not only triggers BMF in FA-deficient mice, but also specifically induces monosomy 7, which is associated with progression to AML in patients with FA, in FA-deficient cells.

Recurrent FAN1 p.W707X Pathogenic Variant Originating Before ad 1800 Underlies High Frequency of Karyomegalic Interstitial Nephritis in South Pacific Islands

Recurrent FAN1 p.W707X Pathogenic Variant Originating Before ad 1800 Underlies High Frequency of Karyomegalic Interstitial Nephritis in South Pacific Islands

Mechanisms of Vertebrate DNA Interstrand Cross-Link Repair.

DNA interstrand cross-links (ICLs) covalently connect the two strands of the double helix and are extremely cytotoxic. Defective ICL repair causes the bone marrow failure and cancer predisposition syndrome, Fanconi anemia, and upregulation of repair causes chemotherapy resistance in cancer. The central event in ICL repair involves resolving the cross-link (unhooking). In this review, we discuss the chemical diversity of ICLs generated by exogenous and endogenous agents. We then describe how proliferating and nonproliferating vertebrate cells unhook ICLs. We emphasize fundamentally new unhooking strategies, dramatic progress in the structural analysis of the Fanconi anemia pathway, and insights into how cells govern the choice between different ICL repair pathways. Throughout, we highlight the many gaps that remain in our knowledge of these fascinating DNA repair pathways.

Identification of Bioactive SNM1A Inhibitors.

SNM1A is a nuclease required to repair DNA interstrand cross-links (ICLs) caused by some anticancer compounds, including cisplatin. Unlike other nucleases involved in ICL repair, SNM1A is not needed to restore other forms of DNA damage. As such, SNM1A is an attractive target for selectively increasing the efficacy of ICL-based chemotherapy. Using a fluorescence-based exonuclease assay, we screened a bioactive library of compounds for inhibition of SNM1A. Of the 52 compounds initially identified as hits, 22 compounds showed dose–response inhibition of SNM1A. An orthogonal gel-based assay further confirmed nine small molecules as SNM1A nuclease activity inhibitors with IC50 values in the mid-nanomolar to low micromolar range. Finally, three compounds showed no toxicity at concentrations able to significantly potentiate the cytotoxicity of cisplatin. These compounds represent potential leads for further optimization to sensitize cells toward chemotherapeutic agents inducing ICL damage.

Targeting CX3CR1 Suppresses the Fanconi Anemia DNA Repair Pathway and Synergizes with Platinum.

Simple SummaryChemotherapeutics exerting their antiproliferative actions by introducing DNA crosslinks, such as platinum drugs, are used to treat numerous cancers. Unfortunately, their therapeutic potential is limited due to adverse side effects and acquired resistance, the latter often associated with enhanced DNA repair capacity. Thus, targeting DNA repair is a promising strategy to lower effective doses and associated side effects, and to restore sensitivity to treatment. The C-X3-C motif chemokine receptor 1 (CX3CR1) is an emerging anticancer target which expression correlates with worse overall survival in cancer patients undergoing DNA damaging treatments. Here we show for the first time that the clinical-phase small molecule inhibitor KAND567 targeting CX3CR1 augments the efficacy of DNA crosslinking chemotherapeutics in cancer cell lines, including platinum resistant models, by interference of the Fanconi anemia DNA repair pathway. Hence, the interplay between CX3CR1 and FA repair provides novel potential therapeutic opportunities in cancers treated with DNA crosslinking agents.The C-X3-C motif chemokine receptor 1 (CX3CR1, fractalkine receptor) is associated with neoplastic transformation, inflammation, neurodegenerative diseases and aging, and the small molecule inhibitor KAND567 targeting CX3CR1 (CX3CR1i) is evaluated in clinical trials for acute systemic inflammation upon SARS-CoV-2 infections. Here we identify a hitherto unknown role of CX3CR1 in Fanconi anemia (FA) pathway mediated repair of DNA interstrand crosslinks (ICLs) in replicating cells. FA pathway activation triggers CX3CR1 nuclear localization which facilitates assembly of the key FA protein FANCD2 into foci. Interfering with CX3CR1 function upon ICL-induction results in inability of replicating cells to progress from S phase, replication fork stalling and impaired chromatin recruitment of key FA pathway factors. Consistent with defective FA repair, CX3CR1i results in increased levels of residual cisplatin-DNA adducts and decreased cell survival. Importantly, CX3CR1i synergizes with platinum agents in a nonreversible manner in proliferation assays including platinum resistant models. Taken together, our results reveal an unanticipated interplay between CX3CR1 and the FA pathway and show for the first time that a clinical-phase small molecule inhibitor targeting CX3CR1 might show benefit in improving responses to DNA crosslinking chemotherapeutics.

Structure of the FA core ubiquitin ligase closing the ID clamp on DNA.

The Fanconi anemia (FA) pathway is essential for the repair of DNA interstrand crosslinks. Central to the pathway is the FA core complex, a ubiquitin ligase of nine subunits that monoubiquitinates the FANCI-FANCD2 (ID) DNA clamp. The 3.1 Å structure of the 1.1-MDa human FA core complex, described here, reveals an asymmetric assembly with two copies of all but the FANCC, FANCE and FANCF subunits. The asymmetry is crucial, as it prevents the binding of a second FANCC-FANCE-FANCF subcomplex that inhibits the recruitment of the UBE2T ubiquitin conjugating enzyme, and instead creates an ID binding site. A single active site then ubiquitinates FANCD2 and FANCI sequentially. We also present the 4.2-Å structures of the human core-UBE2T-ID-DNA complex in three conformations captured during monoubiquitination. They reveal the core-UBE2T complex remodeling the ID-DNA complex, closing the clamp on the DNA before ubiquitination. Monoubiquitination then prevents clamp opening after release from the core.

Current and emerging roles of Cockayne syndrome group B (CSB) protein.

Cockayne syndrome (CS) is a segmental premature aging syndrome caused primarily by defects in the CSA or CSB genes. In addition to premature aging, CS patients typically exhibit microcephaly, progressive mental and sensorial retardation and cutaneous photosensitivity. Defects in the CSB gene were initially thought to primarily impair transcription-coupled nucleotide excision repair (TC-NER), predicting a relatively consistent phenotype among CS patients. In contrast, the phenotypes of CS patients are pleiotropic and variable. The latter is consistent with recent work that implicates CSB in multiple cellular systems and pathways, including DNA base excision repair, interstrand cross-link repair, transcription, chromatin remodeling, RNAPII processing, nucleolin regulation, rDNA transcription, redox homeostasis, and mitochondrial function. The discovery of additional functions for CSB could potentially explain the many clinical phenotypes of CSB patients. This review focuses on the diverse roles played by CSB in cellular pathways that enhance genome stability, providing insight into the molecular features of this complex premature aging disease.

DNA Repair in Huntington's Disease and Spinocerebellar Ataxias: Somatic Instability and Alternative Hypotheses.

The use of genome wide association studies (GWAS) in Huntington’s disease (HD) research, driven by unbiased human data analysis, has transformed the focus of new targets that could affect age at onset. While there is a significant depth of information on DNA damage repair, with many drugs and drug targets, most of this development has taken place in the context of cancer therapy. DNA damage repair in neurons does not rely on DNA replication correction mechanisms. However, there is a strong connection between DNA repair and neuronal metabolism, mediated by nucleotide salvaging and the poly ADP-ribose (PAR) response, and this connection has been implicated in other age-onset neurodegenerative diseases. Validation of leads including the mismatch repair protein MSH3, and interstrand cross-link repair protein FAN1, suggest the mechanism is driven by somatic CAG instability, which is supported by the protective effect of CAA substitutions in the CAG tract. We currently do not understand: how somatic instability is triggered; the state of DNA damage within expanding alleles in the brain; whether this damage induces mismatch repair and interstrand cross-link pathways; whether instability mediates toxicity, and how this relates to human ageing. We discuss DNA damage pathways uncovered by HD GWAS, known roles of other polyglutamine disease proteins in DNA damage repair, and a panel of hypotheses for pathogenic mechanisms.

The interplay of UBE2T and Mule in regulating Wnt/\u03b2-catenin activation to promote hepatocellular carcinoma progression

Emerging evidence indicates the role of cancer stem cells (CSCs) in tumor relapse and therapeutic resistance in patients with hepatocellular carcinoma (HCC). To identify novel targets against liver CSCs, an integrative analysis of publicly available datasets involving HCC clinical and stemness-related data was employed to select genes that play crucial roles in HCC via regulation of liver CSCs. We revealed an enrichment of an interstrand cross-link repair pathway, in which ubiquitin-conjugating enzyme E2 T (UBE2T) was the most significantly upregulated. Consistently, our data showed that UBE2T was upregulated in enriched liver CSC populations. Clinically, UBE2T overexpression in HCC was further confirmed at mRNA and protein levels and was correlated with advanced tumor stage and poor patient survival. UBE2T was found to be critically involved in the regulation of liver CSCs, as evidenced by increases in self-renewal, drug resistance, tumorigenicity, and metastasis abilities. Mule, an E3 ubiquitin ligase, was identified to be the direct protein binding partner of UBE2T. Rather than the canonical role of acting as a mediator to transfer ubiquitin to E3 ligases, UBE2T is surprisingly able to physically bind and regulate the protein expression of Mule via ubiquitination. Mule was found to directly degrade β-catenin protein, and UBE2T was found to mediate liver CSC functions through direct regulation of Mule-mediated β-catenin degradation; this effect was abolished when the E2 activity of UBE2T was impaired. In conclusion, we revealed a novel UBE2T/Mule/β-catenin signaling cascade that is involved in the regulation of liver CSCs, which provides an attractive potential therapeutic target for HCC.

SLFN11 promotes stalled fork degradation that underlies the phenotype in Fanconi anemia cells

AbstractFanconi anemia (FA) is a hereditary disorder caused by mutations in any 1 of 22 FA genes. The disease is characterized by hypersensitivity to interstrand crosslink (ICL) inducers such as mitomycin C (MMC). In addition to promoting ICL repair, FA proteins such as RAD51, BRCA2, or FANCD2 protect stalled replication forks from nucleolytic degradation during replication stress, which may have a profound impact on FA pathophysiology. Recent studies showed that expression of the putative DNA/RNA helicase SLFN11 in cancer cells correlates with cell death on chemotherapeutic treatment. However, the underlying mechanisms of SLFN11-mediated DNA damage sensitivity remain unclear. Because SLFN11 expression is high in hematopoietic stem cells, we hypothesized that SLFN11 depletion might ameliorate the phenotypes of FA cells. Here we report that SLFN11 knockdown in the FA patient-derived FANCD2-deficient PD20 cell line improved cell survival on treatment with ICL inducers. FANCD2−/−SLFN11−/− HAP1 cells also displayed phenotypic rescue, including reduced levels of MMC-induced chromosome breakage compared with FANCD2−/− cells. Importantly, we found that SLFN11 promotes extensive fork degradation in FANCD2−/− cells. The degradation process is mediated by the nucleases MRE11 or DNA2 and depends on the SLFN11 ATPase activity. This observation was accompanied by an increased RAD51 binding at stalled forks, consistent with the role of RAD51 antagonizing nuclease recruitment and subsequent fork degradation. Suppression of SLFN11 protects nascent DNA tracts even in wild-type cells. We conclude that SLFN11 destabilizes stalled replication forks, and this function may contribute to the attrition of hematopoietic stem cells in FA.

M6 A deposition is regulated by PRMT1-mediated arginine methylation of METTL14 in its disordered C-terminal region.

The N6-methyladenosine (m6 A) RNA modification serves crucial functions in RNA metabolism; however, the molecular mechanisms underlying the regulation of m6 A are not well understood. Here, we establish arginine methylation of METTL14, a component of the m6 A methyltransferase complex, as a novel pathway that controls m6 A deposition in mammalian cells. Specifically, protein arginine methyltransferase 1 (PRMT1) interacts with, and methylates the intrinsically disordered C terminus of METTL14, which promotes its interaction with RNA substrates, enhances its RNA methylation activity, and is crucial for its interaction with RNA polymerase II (RNAPII). Mouse embryonic stem cells (mESCs) expressing arginine methylation-deficient METTL14 exhibit significantly reduced global m6 A levels. Transcriptome-wide m6 A analysis identified 1,701 METTL14 arginine methylation-dependent m6 A sites located in 1,290 genes involved in various cellular processes, including stem cell maintenance and DNA repair. These arginine methylation-dependent m6 A sites are associated with enhanced translation of genes essential for the repair of DNA interstrand crosslinks; thus, METTL14 arginine methylation-deficient mESCs are hypersensitive to DNA crosslinking agents. Collectively, these findings reveal important aspects of m6 A regulation and new functions of arginine methylation in RNA metabolism.

NCAPH Recruits and Stabilizes GEN1 in Chromatin to Facilitate DNA Interstrand Crosslink Repair

Repairing damaged DNA and removing all physical connections between sister chromosomes is important for proper chromosomal segregation by contributing to chromosomal stability. Here, we show that depletion of NCAPH exacerbates chromosome segregation errors and cytokinesis failure due to sister-chromatid intertwinement, which is distinct form the ultrafine DNA bridges (UFB) induced by DNA inter-strand crosslinks (DNA-ICL). Importantly, we identified an interaction between NCAPH and GEN1 in the chromatin via binding at the N-terminus of NCAPH. DNA-ICL activation, using ICL-inducing agents, increased the expression and interaction between NCAPH and GEN1 in the soluble nuclear and chromatin fractions, indicating that the NCAPH-GEN1 interaction participates in repairing DNA-ICLs. Moreover, NCAPH recruits and stabilizes GEN1 within chromatin at the G2/M phase, and is associated with DNA-ICL induced damage repair. Therefore, NCAPH-GEN1 specifically responds to DNA-ICLs, and NCAPH recruits and stabilizes GEN1 to ensure DNA damage repair and proper chromosome separation.

Chromosome Instability in Fanconi Anemia: From Breaks to Phenotypic Consequences.

Fanconi anemia (FA), a chromosomal instability syndrome, is caused by inherited pathogenic variants in any of 22 FANC genes, which cooperate in the FA/BRCA pathway. This pathway regulates the repair of DNA interstrand crosslinks (ICLs) through homologous recombination. In FA proper repair of ICLs is impaired and accumulation of toxic DNA double strand breaks occurs. To repair this type of DNA damage, FA cells activate alternative error-prone DNA repair pathways, which may lead to the formation of gross structural chromosome aberrations of which radial figures are the hallmark of FA, and their segregation during cell division are the origin of subsequent aberrations such as translocations, dicentrics and acentric fragments. The deficiency in DNA repair has pleiotropic consequences in the phenotype of patients with FA, including developmental alterations, bone marrow failure and an extreme risk to develop cancer. The mechanisms leading to the physical abnormalities during embryonic development have not been clearly elucidated, however FA has features of premature aging with chronic inflammation mediated by pro-inflammatory cytokines, which results in tissue attrition, selection of malignant clones and cancer onset. Moreover, chromosomal instability and cell death are not exclusive of the somatic compartment, they also affect germinal cells, as evidenced by the infertility observed in patients with FA.