Fanconi Anemia Core Complex Research Articles

Aberrant FAM135B attenuates the efficacy of chemotherapy in colorectal cancer by modulating SRSF1-mediated alternative splicing.

Oxaliplatin is the frontline chemotherapy drug for the treatment of colorectal cancer (CRC) and its insensitivity is a major limitation on therapeutic efficacy. Genomic instability is the prominent feature of CRC and is considered to correlate with response to treatments. However, the underlying mechanism of insensitivity to oxaliplatin (L-OHP) remains largely unclear. Herein, sequence similarity 135 family member B (FAM135B) is identified as a frequently mutated gene in CRC and is critical for CRC proliferation and impaired response to L-OHP by controlling SRSF1-mediated alternative splicing. Specifically, FAM135B promotes the nuclear translocation of SRSF1 by synergistically binding with SRPK1 and regulates SRSF1-mediated splicing of DNA repair genes. FAM135B-induced exon IV inclusion of FAAP20 mediates its binding with FACNA and enhances the functional integrity of the FA core complex, thereby activating the FA pathway and resulting in inter-strand crosslink (ICL) lesion repair and L-OHP insensitivity. These findings reveal that the FAM135B-SRSF1 axis-mediated splicing contributes to DNA repair and chemotherapeutic insensitivity in CRC. Targeting FAM135B represents a potential strategy for CRC treatment.

The Fanconi anemia pathway induces chromothripsis and ecDNA-driven cancer drug resistance

Chromothripsis describes the catastrophic shattering of mis-segregated chromosomes trapped within micronuclei. Although micronuclei accumulate DNA double-strand breaks and replication defects throughout interphase, how chromosomes undergo shattering remains unresolved. Using CRISPR-Cas9 screens, we identify a non-canonical role of the Fanconi anemia (FA) pathway as a driver of chromothripsis. Inactivation of the FA pathway suppresses chromosome shattering during mitosis without impacting interphase-associated defects within micronuclei. Mono-ubiquitination of FANCI-FANCD2 by the FA core complex promotes its mitotic engagement with under-replicated micronuclear chromosomes. The structure-selective SLX4-XPF-ERCC1 endonuclease subsequently induces large-scale nucleolytic cleavage of persistent DNA replication intermediates, which stimulates POLD3-dependent mitotic DNA synthesis to prime shattered fragments for reassembly in the ensuing cell cycle. Notably, FA-pathway-induced chromothripsis generates complex genomic rearrangements and extrachromosomal DNA that confer acquired resistance to anti-cancer therapies. Our findings demonstrate how pathological activation of a central DNA repair mechanism paradoxically triggers cancer genome evolution through chromothripsis.

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.

Research progress of the Fanconi anemia pathway and premature ovarian insufficiency†.

The Fanconi anemia pathway is a key pathway involved in the repair of deoxyribonucleic acidinterstrand crosslinking damage, which chiefly includes the following four modules: lesion recognition, Fanconi anemia core complex recruitment, FANCD2-FANCI complex monoubiquitination, and downstream events (nucleolytic incision, translesion synthesis, and homologous recombination). Mutations or deletions of multiple Fanconi anemia genes in this pathway can damage the interstrand crosslinking repair pathway and disrupt primordial germ cell development and oocyte meiosis, thereby leading to abnormal follicular development. Premature ovarian insufficiency is a gynecological clinical syndrome characterized by amenorrhea and decreased fertility due to decreased oocyte pool, accelerated follicle atresia, and loss of ovarian function in women <40years old. Furthermore, in recent years, several studies have detected mutations in the Fanconi anemia gene in patients with premature ovarian insufficiency. In addition, some patients with Fanconi anemia exhibit symptoms of premature ovarian insufficiency and infertility. The Fanconi anemia pathway and premature ovarian insufficiency are closely associated.

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.

FAAP100 is required for the resolution of transcription-replication conflicts in primordial germ cells

BackgroundThe maintenance of genome stability in primordial germ cells (PGCs) is crucial for the faithful transmission of genetic information and the establishment of reproductive reserve. Numerous studies in recent decades have linked the Fanconi anemia (FA) pathway with fertility, particularly PGC development. However, the role of FAAP100, an essential component of the FA core complex, in germ cell development is unexplored.ResultsWe find that FAAP100 plays an essential role in R-loop resolution and replication fork protection to counteract transcription-replication conflicts (TRCs) during mouse PGC proliferation. FAAP100 deletion leads to FA pathway inactivation, increases TRCs as well as cotranscriptional R-loops, and contributes to the collapse of replication forks and the generation of DNA damage. Then, the activated p53 signaling pathway triggers PGC proliferation defects, ultimately resulting in insufficient establishment of reproductive reserve in both sexes of mice.ConclusionsOur findings suggest that FAAP100 is required for the resolution of TRCs in PGCs to safeguard their genome stability.

Pan-cancer analysis of the prognostic and immunological role of Fanconi anemia complementation group E.

Fanconi anemia (FA) genes contribute to tumorigenesis by regulating DNA repair. Despite its importance for assembly and functionality of the FA core complex, no pan-cancer analysis of FANCE was performed. We aimed to provide a comprehensive understanding of the role of FANCE in cancers. Based on The Cancer Genome Atlas (TCGA), Cancer Cell Line Encyclopedia (CCLE), Genotype Tissue-Expression (GTEx), Human Protein Atlas (HPA), Gene Expression Omnibus (GEO), and Cancer Single-cell Atlas (CancerSEA) databases, we investigated the carcinogenicity of FANCE using various bioinformatics methods, including FANCE expression and prognosis, immune invasion, tumor mutation burden, microsatellite instability, and neoantigens. We monitored Fance mutations in mice that caused tumorigenesis. FANCE expression and activity scores were upregulated in 15 and 21 cancers. High expression of FANCE affected shorter overall survival (OS) in seven cancers and longer overall survival in three cancers. It was correlated with shorter overall survival and progression-free interval (PFI) in endometrial cancer and longer overall survival and PFI in cervical cancer. FANCE expression negatively correlated with stromal/immune scores in 21 cancers including cervical cancer, endometrial cancer, and ovarian cancer. FANCE expression negatively correlated with CD8 T cells in endometrial cancer and positively correlated with M1 macrophages in cervical cancer, possibly related to cancer prognosis. FANCE positively correlated with immune checkpoint inhibitors PD-1, PD-L1, and CTLA4 in endometrial cancer and ovarian cancer. FANCE expression positively correlated with microsatellite instability, tumor mutational burden, and neoantigens in 7, 22, and five cancers, especially in endometrial cancer, potentially increasing the effectiveness of immunotherapy. Single-cell sequencing data showed FANCE was primarily expressed in cancer cells in cervical and ovarian cancer, and in fibroblasts in endometrial cancer. Fance heterozygous mutant mice had increased tumor incidences and shorter overall survival and tumor-free survival (TFS) than Fance homozygous mutant mice and wild-type mice. Conclusively, FANCE potential to serve as a biomarker for cancer prognosis and may predict cancer immunotherapy responses. Fance heterozygous mutant resulted in increased tumorigenesis and poor prognosis in mice.

The DNA-damage kinase ATR activates the FANCD2-FANCI clamp by priming it for ubiquitination.

DNA interstrand cross-links are tumor-inducing lesions that block DNA replication and transcription. When cross-links are detected at stalled replication forks, ATR kinase phosphorylates FANCI, which stimulates monoubiquitination of the FANCD2-FANCI clamp by the Fanconi anemia core complex. Monoubiquitinated FANCD2-FANCI is locked onto DNA and recruits nucleases that mediate DNA repair. However, it remains unclear how phosphorylation activates this pathway. Here, we report structures of FANCD2-FANCI complexes containing phosphomimetic FANCI. We observe that, unlike wild-type FANCD2-FANCI, the phosphomimetic complex closes around DNA, independent of the Fanconi anemia core complex. The phosphomimetic mutations do not substantially alter DNA binding but instead destabilize the open state of FANCD2-FANCI and alter its conformational dynamics. Overall, our results demonstrate that phosphorylation primes the FANCD2-FANCI clamp for ubiquitination, showing how multiple posttranslational modifications are coordinated to control DNA repair.

FANCL supports Parkin-mediated mitophagy in a ubiquitin ligase-independent manner

Fanconi anemia (FA) is the most common inherited bone marrow failure syndrome. The FA proteins have functions in genome maintenance and in the cytoplasmic process of selective autophagy, beyond their canonical roles of repairing DNA interstrand cross-links. FA core complex proteins FANCC, FANCF, FANCL, FANCA, FANCD2, BRCA1 and BRCA2, which previously had no known direct functions outside the nucleus, have recently been implicated in mitophagy. Although mutations in FANCL account for only a very small number of cases in FA families, it plays a key role in the FA pathophysiology and might drive carcinogenesis. Here, we demonstrate that FANCL protein is present in mitochondria in the control and Oligomycin and Antimycin (OA)-treated cells and its ubiquitin ligase activity is not required for its localization to mitochondria. CRISPR/Cas9-mediated knockout of FANCL in HeLa cells overexpressing parkin results in increased sensitivity to mitochondrial stress and defective clearing of damaged mitochondria upon OA treatment. This defect was reversed by the reintroduction of either wild-type FANCL or FANCL(C307A), a mutant lacking ubiquitin ligase activity. To summarize, FANCL protects from mitochondrial stress and supports Parkin-mediated mitophagy in a ubiquitin ligase-independent manner.

Fancb deficiency causes premature ovarian insufficiency in mice

Fanconi anemia complementation group B (FANCB) protein is a major component of the Fanconi anemia (FA) core complex and plays an important role in hematopoiesis and germ cell development. Deletion of Fancb gene causes the defect of primordial germ cell (PGC) development and infertility in male mice. However, it remains unknown whether Fancb is required for female germ cell development. In this study, we found that the fertility of Fancb knockout male mice in C57/ICR mixed backgrounds was not affected. Female Fancb-/- mice were obtained by crossing Fancb+/- females with Fancb-/Y males. The number of PGCs was dramatically decreased in Fancb-/- females. Very few oocytes were observed after birth and the primordial follicle pool was completely depleted at 6weeks of age in Fancb-/- females. However, the remained oocytes from Fancb-/- mice were normal in fertilization and embryonic development from 2-cell to the blastocyst stage. We also found that Fancb and Fancl double-knockout males were also fertile and the number of sperm in epididymis was not reduced as compared to that of Fancb-/- and Fancl-/- single-knockout mice. Taken together, these results showed that Fancb is also essential for female germ cell development. Inactivation of Fancb causes massive germ cell loss and infertility in adult females. We also found that Fancb and Fancl do not act synergistically in regulating germ cell development.

Novel Bi-Allelic Variants of FANCM Cause Sertoli Cell-Only Syndrome and Non-Obstructive Azoospermia

Non-obstructive azoospermia (NOA) is the most severe disease in male infertility, but the genetic causes for the majority of NOA remain unknown. FANCM is a member of Fanconi Anemia (FA) core complex, whose defects are associated with cell hypersensitivity to DNA interstrand crosslink (ICL)-inducing agents. It was reported that variants in FANCM (MIM: 609644) might cause azoospermia or oligospermia. However, there is still a lack of evidence to explain the association between different FANCM variants and male infertility phenotypes. Herein, we identified compound heterozygous variants in FANCM in two NOA-affected brothers (c. 1778delG:p. R593Qfs*76 and c. 1663G > T:p. V555F), and a homozygous variant in FANCM (c. 1972C > T:p. R658X) in a sporadic case with NOA, respectively. H&E staining and immunohistochemistry showed Sertoli cell-only Syndrome (SCOS) in the three patients with NOA. Collectively, our study expands the knowledge of variants in FANCM, and provides a new insight to understand the genetic etiology of NOA.

Novel Ubiquitinated Proteins Downstream of the Fanconi Anemia Core Complex

The Fanconi anemia (FA) pathway is a major player in the control of DNA replication integrity in response to replication stress. Germline defect in the pathway results in the FA syndrome characterized by developmental abnormalities, bone marrow (BM) failure, and genome instability which greatly elevates the incidence of cancers. A pivotal step in the activation of the FA DNA repair pathway is the monoubiquitination of the FANCD2 and FANCI proteins (ID2) by the FA core complex, a unique ubiquitin ligase complex which includes eight proteins (FANCA-FANCG, FANCL, and FAAP100) and UBE2T/FANCT. This monoubiquitination event enables the recruitment of the ID2 complex to chromatin and nuclear foci at sites of DNA damage. Cells with mutations in any of the FA core complex proteins lack the ability to monoubiquitinated ID2, making ID2 ubiquitination a convergence point in the pathway, with an estimation of>90% FA patients defective in this step. Additionally, somatic mutations In FA genes render tumor cells sensitive to DNA crosslinking agents, so identification of FA pathway defects provides an opportunity for therapeutic targeting. In search for additional potential target/substrate of this unique FA core ubiquitin ligase complex, we performed a high throughput genome-wide ubiquitin-specific proteomics (UbiScan) screen and found, in addition to the ID2 complex, many ubiquitinated proteins are dysregulated (mostly downregulated) in FA deficient cells compared with that of FA proficient cells.We used a Ubiquitin Remnant Motif (K- ∑-GG) Antibody Bead Conjugate (Cell Signaling Technology), a proprietary ubiquitin branch (“K- ∑-GG”) antibody with specificity for a di-glycine tag that is the remnant of ubiquitin left on protein substrates after trypsin digestion, to enrich ubiquitinated peptides from trypsin digested cell samples (shNT vs shFANCA). This enrichment is followed by LC-MS/MS analysis for quantitative profiles of hundreds to over a thousand nonredundant ubiquitinated sequences. We were successful in demonstrating that under steady-state conditions (without proteasome inhibitor treatment), the ubiquitinated forms of both FANCD2 and FANCI proteins are much higher in control (shNT) HeLa cells compared with that of the cells depleted of FANCA (shFANCA).We then collaborated with the Cell signaling technology to perform a high throughput UbiScan® analysis of total ubiquitinated proteins both in total nuclei and chromatin fractions under replicative stress conditions. UbiScan® enables researchers to isolate, identify and quantitate large numbers of ubiquitin-modified cellular peptides with a high degree of specificity and sensitivity, providing a global overview of the ubiquitination sites in cellular proteins in cell and tissue samples without preconceived biases about where these modified sites occur. A total of 16,249 redundant modified peptide assignments to 7,856 modified sites for the Ubiquitin K-GG Remnant Motif Antibody were obtained. As expected, the amount of monoubiquitinated FANCD2 (at K651) and FANCI (at K523) were highly reduced in both the nuclear and chromatin fractions of Hela cells depleted of FANCA (shA). Consistent with the earlier findings, the amount of ubiquitinated ID2 proteins were extremely low in the chromatin fraction of the Hela cells depleted of FANCA.Since there are numerous ubiquitinated proteins found to be dysregulated in our UbiScan analyses, we used the following criteria to select the target proteins based on; a) -fold changes, and b) proteins that are known to participate in the DNA repair signaling pathways. We validated our UbiScan results by using an assay system to detect endogenous protein ubiquitination. We also found a significant reduction in the ubiquitination of several DNA repair-related proteins (found in our UbiScan analysis) in FANCA deficient cells. To assess FA pathway functions, we generated HAP1 and appropriate cells knock out of these select ubiquitinated target proteins by using CRISPR-Cas9 system. Then, the KO cells were examined for FA pathway functions. These results will be discussed.In conclusion, our findings reveal that the FA core ubiquitin ligase complex regulates (directly or indirectly) the ubiquitinated levels of many novel proteins outside of the ID2 complex, and these novel target proteins may provide important additional mechanistic insights into the FA DNA repair pathway. DisclosuresNo relevant conflicts of interest to declare.

Abnormal migration behavior linked to Rac1 signaling contributes to primordial germ cell exhaustion in Fanconi anemia pathway-deficient Fancg-/- embryos.

Fanconi anemia (FA) is a rare human genetic disorder characterized by bone marrow failure, predisposition to cancer and developmental defects including hypogonadism. Reproductive defects leading to germ cell aplasia are the most consistent phenotypes seen in FA mouse models. We examined the role of the nuclear FA core complex gene Fancg in the development of primordial germ cells (PGCs), the embryonic precursors of adult gametes, during fetal development. PGC maintenance was severely impaired in Fancg−/− embryos. We observed a defect in the number of PGCs starting at E9.5 and a strong attrition at E11.5 and E13.5. Remarkably, we observed a mosaic pattern reflecting a portion of testicular cords devoid of PGCs in E13.5 fetal gonads. Our in vitro and in vivo data highlight a potential role of Fancg in the proliferation and in the intrinsic cell motility abilities of PGCs. The random migratory process is abnormally activated in Fancg−/− PGCs, altering the migration of cells. Increased cell death and PGC attrition observed in E11.5 Fancg−/− embryos are features consistent with delayed migration of PGCs along the migratory pathway to the genital ridges. Moreover, we show that an inhibitor of RAC1 mitigates the abnormal migratory pattern observed in Fancg−/− PGCs.

FANCI functions as a repair/apoptosis switch in response to DNA crosslinks.

Cells counter DNA damage through repair or apoptosis, yet a direct mechanism for this choice has remained elusive. When facing interstrand crosslinks (ICLs), the ICL-repair protein FANCI heterodimerizes with FANCD2 to initiate ICL excision. We found that FANCI alternatively interacts with a pro-apoptotic factor, PIDD1, to enable PIDDosome (PIDD1-RAIDD-caspase-2) formation and apoptotic death. FANCI switches from FANCD2/repair to PIDD1/apoptosis signaling in the event of ICL-repair failure. Specifically, removing key endonucleases downstream of FANCI/FANCD2, increasing ICL levels, or allowing damaged cells into mitosis (when repair is suppressed) all suffice for switching. Reciprocally, apoptosis-committed FANCI reverts from PIDD1 to FANCD2 after a failed attempt to assemble the PIDDosome. Monoubiquitination and deubiquitination at FANCI K523 impact interactor selection. These data unveil a repair-or-apoptosis switch in eukaryotes. Beyond ensuring the removal of unrepaired genomes, the switch's bidirectionality reveals that damaged cells can offset apoptotic defects via de novo attempts at lesion repair.

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.

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).

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.

The ubiquitination machinery of the Fanconi Anemia DNA repair pathway

The Fanconi Anemia (FA) pathway maintains genome stability by preventing DNA damage from occurring when replication is blocked. Central to the FA pathway is the monoubiquitination of FANCI-FANCD2 mediated by a ubiquitin RING-E3 ligase complex called the FA core complex. Genetic mutation in any component of the FA core complex results in defective FANCI-FANCD2 monoubiquitination and phenotypes of DNA damage sensitivity, birth defects, early-onset bone marrow failure and cancer. Here, we discuss the mechanisms of the FA core complex and FANCI-FANCD2 monoubiquitination at sites of blocked replication and review our current understanding of the biological functions of these proteins in replication fork protection.

Acetylation modulates the Fanconi anemia pathway by protecting FAAP20 from ubiquitin-mediated proteasomal degradation

Fanconi anemia (FA) is a chromosome instability syndrome of children caused by inherited mutations in one of FA genes, which together constitute a DNA interstrand cross-link (ICL) repair, or the FA pathway. Monoubiquitination of Fanconi anemia group D2 protein (FANCD2) by the multisubunit ubiquitin E3 ligase, the FA core complex, is an obligate step in activation of the FA pathway, and its activity needs to be tightly regulated. FAAP20 is a key structural component of the FA core complex, and regulated proteolysis of FAAP20 mediated by prolyl cis-trans isomerization and phosphorylation at a consensus phosphodegron motif is essential for preserving the integrity of the FA core complex, and thus FANCD2 monoubiquitination. However, how ubiquitin-dependent FAAP20 degradation is modulated to fine-tune FA pathway activation remains largely un-known. Here, we present evidence that FAAP20 is acetylated by the acetyltransferase p300/CBP on lysine 152, the key residue that when polyubiquitinated results in the degradation of FAAP20. Acetylation or mutation of the lysine residue stabilizes FAAP20 by preventing its ubiquitination, thereby protecting it from proteasome-dependent FAAP20 degradation. Consequently, disruption of the FAAP20 acetylation pathway impairs FANCD2 activation. Together, our study reveals a competition mechanism between ubiquitination and acetylation of a common lysine residue that controls FAAP20 stability and highlights a complex balancing between different posttranslational modifications as a way to refine the FA pathway signaling required for DNA ICL repair and genome stability.

Deep learning enables the atomic structure determination of the Fanconi Anemia core complex from cryoEM.

Cryo-electron microscopy of protein complexes often leads to moderate resolution maps (4-8 Å), with visible secondary-structure elements but poorly resolved loops, making model building challenging. In the absence of high-resolution structures of homologues, only coarse-grained structural features are typically inferred from these maps, and it is often impossible to assign specific regions of density to individual protein subunits. This paper describes a new method for overcoming these difficulties that integrates predicted residue distance distributions from a deep-learned convolutional neural network, computational protein folding using Rosetta, and automated EM-map-guided complex assembly. We apply this method to a 4.6 Å resolution cryoEM map of Fanconi Anemia core complex (FAcc), an E3 ubiquitin ligase required for DNA interstrand crosslink repair, which was previously challenging to interpret as it comprises 6557 residues, only 1897 of which are covered by homology models. In the published model built from this map, only 387 residues could be assigned to the specific subunits with confidence. By building and placing into density 42 deep-learning-guided models containing 4795 residues not included in the previously published structure, we are able to determine an almost-complete atomic model of FAcc, in which 5182 of the 6557 residues were placed. The resulting model is consistent with previously published biochemical data, and facilitates interpretation of disease-related mutational data. We anticipate that our approach will be broadly useful for cryoEM structure determination of large complexes containing many subunits for which there are no homologues of known structure.