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SMC5/6 acts jointly with Fanconi anemia factors to support DNA repair and genome stability

ABSTRACT: SMC5/6 function in genome integrity remains elusive. Here we show that SMC5 dysfunction in avian DT40 B cells causes mitotic delay and hypersensitivity towards DNA intra- and inter-strand crosslinkers (ICLs), with smc5 mutants being epistatic to FANCC and FANCM mutations affecting the Fanconi anemia (FA) pathway. Mutations in the checkpoint clamp loader RAD17 and the DNA helicase DDX11, acting in an FA-like pathway, do not aggravate the damage sensitivity caused by SMC5 dysfunction in DT40 cells. SMC5/6 knockdown in HeLa cells causes MMC sensitivity, increases nuclear bridges, micronuclei and mitotic catastrophes in a manner similar and epistatic to FANCD2 knockdown. In both DT40 and HeLa systems, SMC5/6 deficiency increases FANCD2 ubiquitylation and, unlike FANCD2 depletion, RAD51 focus formation. SMC5/6 components further physically interact with FANCD2-I in human cells. Altogether, our data suggest that SMC5/6 function jointly with the FA pathway to support genome integrity and DNA repair and may be implicated in FA or FA-related human disorders.

SUBMITTER: Dr. Francesco Rossi

PROVIDER: S-SCDT-EMBOR-2019-48222V1 | biostudies-other |

REPOSITORIES: biostudies-other

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SMC5/6 acts jointly with Fanconi anemia factors to support DNA repair and genome stability.

Project description:SMC5/6 function in genome integrity remains elusive. Here, we show that SMC5 dysfunction in avian DT40 B cells causes mitotic delay and hypersensitivity toward DNA intra- and inter-strand crosslinkers (ICLs), with smc5 mutants being epistatic to FANCC and FANCM mutations affecting the Fanconi anemia (FA) pathway. Mutations in the checkpoint clamp loader RAD17 and the DNA helicase DDX11, acting in an FA-like pathway, do not aggravate the damage sensitivity caused by SMC5 dysfunction in DT40 cells. SMC5/6 knockdown in HeLa cells causes MMC sensitivity, increases nuclear bridges, micronuclei, and mitotic catastrophes in a manner similar and non-additive to FANCD2 knockdown. In both DT40 and HeLa systems, SMC5/6 deficiency does not affect FANCD2 ubiquitylation and, unlike FANCD2 depletion, RAD51 focus formation. SMC5/6 components further physically interact with FANCD2-I in human cells. Altogether, our data suggest that SMC5/6 functions jointly with the FA pathway to support genome integrity and DNA repair and may be implicated in FA or FA-related human disorders.

| S-EPMC7001510 | biostudies-literature

The Fanconi Anemia Pathway Maintains Genome Stability by Coordinating Replication and Transcription.

Project description:DNA replication stress can cause chromosomal instability and tumor progression. One key pathway that counteracts replication stress and promotes faithful DNA replication consists of the Fanconi anemia (FA) proteins. However, how these proteins limit replication stress remains largely elusive. Here we show that conflicts between replication and transcription activate the FA pathway. Inhibition of transcription or enzymatic degradation of transcription-associated R-loops (DNA:RNA hybrids) suppresses replication fork arrest and DNA damage occurring in the absence of a functional FA pathway. Furthermore, we show that simple aldehydes, known to cause leukemia in FA-deficient mice, induce DNA:RNA hybrids in FA-depleted cells. Finally, we demonstrate that the molecular mechanism by which the FA pathway limits R-loop accumulation requires FANCM translocase activity. Failure to activate a response to physiologically occurring DNA:RNA hybrids may critically contribute to the heightened cancer predisposition and bone marrow failure of individuals with mutated FA proteins.

| S-EPMC4644232 | biostudies-literature

Human Fanconi anemia monoubiquitination pathway promotes homologous DNA repair.

Project description:Fanconi anemia (FA) is a recessive disorder characterized by congenital abnormalities, progressive bone-marrow failure, and cancer susceptibility. Cells from FA patients are hypersensitive to agents that produce DNA crosslinks and, after treatment with these agents, have pronounced chromosome breakage and other cytogenetic abnormalities. Eight FANC genes have been cloned, and the encoded proteins interact in a common cellular pathway. DNA-damaging agents activate the monoubiquitination of FANCD2, resulting in its targeting to nuclear foci that also contain BRCA1 and BRCA2/FANCD1, proteins involved in homology-directed DNA repair. Given the interaction of the FANC proteins with BRCA1 and BRCA2, we tested whether cells from FA patients (groups A, G, and D2) and mouse Fanca-/- cells with a targeted mutation are impaired for this repair pathway. We find that both the upstream (FANCA and FANCG) and downstream (FANCD2) FA pathway components promote homology-directed repair of chromosomal double-strand breaks (DSBs). The FANCD2 monoubiquitination site is critical for normal levels of repair, whereas the ATM phosphorylation site is not. The defect in these cells, however, is mild, differentiating them from BRCA1 and BRCA2 mutant cells. Surprisingly, we provide evidence that these proteins, like BRCA1 but unlike BRCA2, promote a second DSB repair pathway involving homology, i.e., single-strand annealing. These results suggest an early role for the FANC proteins in homologous DSB repair pathway choice.

| S-EPMC545844 | biostudies-other

Mutated Fanconi anemia pathway in non-Fanconi anemia cancers.

Project description:An extremely high cancer incidence and the hypersensitivity to DNA crosslinking agents associated with Fanconi Anemia (FA) have marked it to be a unique genetic model system to study human cancer etiology and treatment, which has emerged an intense area of investigation in cancer research. However, there is limited information about the relationship between the mutated FA pathway and the cancer development or/and treatment in patients without FA. Here we analyzed the mutation rates of the seventeen FA genes in 68 DNA sequence datasets. We found that the FA pathway is frequently mutated across a variety of human cancers, with a rate mostly in the range of 15 to 35 % in human lung, brain, bladder, ovarian, breast cancers, or others. Furthermore, we found a statistically significant correlation (p < 0.05) between the mutated FA pathway and the development of human bladder cancer that we only further analyzed. Together, our study demonstrates a previously unknown fact that the mutated FA pathway frequently occurs during the development of non-FA human cancers, holding profound implications directly in advancing our understanding of human tumorigenesis as well as tumor sensitivity/resistance to crosslinking drug-relevant chemotherapy.

| S-EPMC4653013 | biostudies-literature

The Fanconi anemia pathway in replication stress and DNA crosslink repair.

Project description:Interstand crosslinks (ICLs) are DNA lesions where the bases of opposing DNA strands are covalently linked, inhibiting critical cellular processes such as transcription and replication. Chemical agents that generate ICLs cause chromosomal abnormalities including breaks, deletions and rearrangements, making them highly genotoxic compounds. This toxicity has proven useful for chemotherapeutic treatment against a wide variety of cancer types. The majority of our understanding of ICL repair in humans has been uncovered through analysis of the rare genetic disorder Fanconi anemia, in which patients are extremely sensitive to crosslinking agents. Here, we discuss recent insights into ICL repair gained using new repair assays and highlight the role of the Fanconi anemia repair pathway during replication stress.

| S-EPMC3890373 | biostudies-literature

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

Project description: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.

| S-EPMC8306909 | biostudies-literature

Fanconi Anemia germline variants as susceptibility factors in aplastic anemia, MDS and AML.

Project description:Using next generation sequencing we have systematically analyzed a large cohort of 489 patients with bone marrow failure (BMF), including myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), aplastic anemia (AA), and related conditions for the presence of germline (GL) alterations in Fanconi Anemia (FA) and telomerase genes. We have detected an increased frequency of heterozygous FA gene mutations in MDS and to lesser degree in AML suggesting that the presence of one normal allele may not be completely protective and indeed heterozygous FA lesions may have a long latency period before hematologic manifestation. In contrast, GL telomerase gene mutations were not associated with increased disease risk. When compared to large control cohorts, we have not detected an increased frequency of damaging variants among telomerase complex genes, including those previously believed to be involved in the pathogenesis of AA. Our results may suggest that while low penetrance and delayed disease onset can confound identification of genetic predisposition factors, GL FA alterations can be also associated with MDS.

| S-EPMC5788620 | biostudies-literature

Genomic signature of Fanconi anemia DNA repair pathway deficiency in cancer

Project description:Fanconi anemia (FA), a model syndrome of genome instability, is caused by a deficiency in DNA interstrand crosslink (ICL) repair resulting in chromosome breakage. The FA repair pathway protects against carcinogenic endogenous and exogenous aldehydes. Individuals with FA are hundreds to thousands-fold more likely to develop head and neck (HNSCC), esophageal and anogenital squamous cell carcinomas (SCCs). Molecular studies of SCCs from individuals with FA (FA SCCs) are limited, and it is unclear how FA SCCs relate to sporadic HNSCCs primarily driven by tobacco and alcohol exposure or human papillomavirus (HPV) infection. Here, by sequencing FA SCCs, we demonstrate that the primary genomic signature of FA-deficiency is the presence of high numbers of structural variants (SVs). SVs are enriched for small deletions, unbalanced translocations, and fold-back inversions, and are often connected, thereby forming complex rearrangements. They arise in the context of TP53 loss, but not HPV infection, and lead to somatic copy number alterations of HNSCC driver genes. We further show that FA pathway deficiency may lead to epithelial-to-mesenchymal transition and enhanced keratinocyte intrinsic inflammatory signaling, which would contribute to the aggressive nature of FA SCCs. We propose that genomic instability in sporadic HPV-negative HNSCC may arise consequent to the FA repair pathway being overwhelmed by ICL damage caused by alcohol and tobacco-derived aldehydes, making FA SCC a powerful model to study tumorigenesis resulting from DNA crosslinking damage.

2022-09-04 | GSE195811 | GEO

The Fanconi anemia pathway promotes replication-dependent DNA interstrand cross-link repair.

Project description:Fanconi anemia is a human cancer predisposition syndrome caused by mutations in 13 Fanc genes. The disorder is characterized by genomic instability and cellular hypersensitivity to chemicals that generate DNA interstrand cross-links (ICLs). A central event in the activation of the Fanconi anemia pathway is the mono-ubiquitylation of the FANCI-FANCD2 complex, but how this complex confers ICL resistance remains enigmatic. Using a cell-free system, we showed that FANCI-FANCD2 is required for replication-coupled ICL repair in S phase. Removal of FANCD2 from extracts inhibits both nucleolytic incisions near the ICL and translesion DNA synthesis past the lesion. Reversal of these defects requires ubiquitylated FANCI-FANCD2. Our results show that multiple steps of the essential S-phase ICL repair mechanism fail when the Fanconi anemia pathway is compromised.

| S-EPMC2909596 | biostudies-literature

Mutations in ERCC4, encoding the DNA-repair endonuclease XPF, cause Fanconi anemia.

Project description:Fanconi anemia (FA) is a rare genomic instability disorder characterized by progressive bone marrow failure and predisposition to cancer. FA-associated gene products are involved in the repair of DNA interstrand crosslinks (ICLs). Fifteen FA-associated genes have been identified, but the genetic basis in some individuals still remains unresolved. Here, we used whole-exome and Sanger sequencing on DNA of unclassified FA individuals and discovered biallelic germline mutations in ERCC4 (XPF), a structure-specific nuclease-encoding gene previously connected to xeroderma pigmentosum and segmental XFE progeroid syndrome. Genetic reversion and wild-type ERCC4 cDNA complemented the phenotype of the FA cell lines, providing genetic evidence that mutations in ERCC4 cause this FA subtype. Further biochemical and functional analysis demonstrated that the identified FA-causing ERCC4 mutations strongly disrupt the function of XPF in DNA ICL repair without severely compromising nucleotide excision repair. Our data show that depending on the type of ERCC4 mutation and the resulting balance between both DNA repair activities, individuals present with one of the three clinically distinct disorders, highlighting the multifunctional nature of the XPF endonuclease in genome stability and human disease.

| S-EPMC3644630 | biostudies-literature

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