Functional relationships of FANCC to homologous recombination, translesion synthesis, and BLM

The BRCA2 breast cancer tumor suppressor is involved in the repair of double strand breaks and broken replication forks by homologous recombination through its interaction with DNA repair protein Rad51. Cells defective in BRCA2.FANCD1 are extremely sensitive to mitomycin C (MMC) similarly to cells deficient in any of the Fanconi anemia (FA) complementation group proteins (FANC). These observations suggest that the FA pathway and the BRCA2 and Rad51 repair pathway may be linked, although a functional connection between these pathways in DNA damage signaling remains to be determined. Here, we systematically investigated the interaction between these pathways. We show that in response to DNA damage, BRCA2-dependent Rad51 nuclear focus formation was normal in the absence of FANCD2 and that FANCD2 nuclear focus formation and mono-ubiquitination appeared normal in BRCA2-deficient cells. We report that the absence of BRCA2 substantially reduced homologous recombination repair of DNA breaks, whereas the absence of FANCD2 had little effect. Furthermore, we established that depletion of BRCA2 or Rad51 had a greater effect on cell survival in response to MMC than depletion of FANCD2 and that depletion of BRCA2 in FANCD2 mutant cells further sensitized these cells to MMC. Our results suggest that FANCD2 mediates double strand DNA break repair independently of Rad51-associated homologous recombination.

Uveal melanoma (UM) is unique among cancers in displaying reduced endogenous levels of sister chromatid exchange (SCE). Here we demonstrate that FANCD2 expression is reduced in UM and that ectopic expression of FANCD2 increased SCE. Similarly, FANCD2-deficient fibroblasts (PD20) derived from Fanconi anaemia patients displayed reduced spontaneous SCE formation relative to their FANCD2-complemented counterparts, suggesting that this observation is not specific to UM. In addition, spontaneous RAD51 foci were reduced in UM and PD20 cells compared with FANCD2-proficient cells. This is consistent with a model where spontaneous SCEs are the end product of endogenous recombination events and implicates FANCD2 in the promotion of recombination-mediated repair of endogenous DNA damage and in SCE formation during normal DNA replication. In both UM and PD20 cells, low SCE was reversed by inhibiting DNA-PKcs (DNA-dependent protein kinase, catalytic subunit). Finally, we demonstrate that both PD20 and UM are sensitive to acetaldehyde, supporting a role for FANCD2 in repair of lesions induced by such endogenous metabolites. Together, these data suggest FANCD2 may promote spontaneous SCE by influencing which double-strand break repair pathway predominates during normal S-phase progression.

Fanconi anemia (FA) is a cancer susceptibility syndrome characterized by sensitivity to DNA-damaging agents. The FA proteins (FANCs) are implicated in DNA repair, although the precise mechanisms by which FANCs process DNA lesions are not fully understood. An epistatic relationship between the FA pathway and translesion synthesis (TLS, a post-replication DNA repair mechanism) has been suggested, but the basis for cross-talk between the FA and TLS pathways is poorly understood. We show here that ectopic overexpression of the E3 ubiquitin ligase Rad18 (a central regulator of TLS) induces DNA damage-independent mono-ubiquitination of proliferating cell nuclear antigen (PCNA) (a known Rad18 substrate) and FANCD2. Conversely, DNA damage-induced mono-ubiquitination of both PCNA and FANCD2 is attenuated in Rad18-deficient cells, demonstrating that Rad18 contributes to activation of the FA pathway. WT Rad18 but not an E3 ubiquitin ligase-deficient Rad18 C28F mutant fully complements both PCNA ubiquitination and FANCD2 activation in Rad18-depleted cells. Rad18-induced mono-ubiquitination of FANCD2 is not observed in FA core complex-deficient cells, demonstrating that Rad18 E3 ligase activity alone is insufficient for FANCD2 ubiquitylation. Instead, Rad18 promotes FA core complex-dependent FANCD2 ubiquitination in a manner that is secondary to PCNA mono-ubiquitination. Taken together, these results demonstrate a novel Rad18-dependent mechanism that couples activation of the FA pathway with TLS.

BLM has been implicated in DNA double-strand break (DSB) repair, but its precise role remains obscure. To explore this, we generated BLM(-/-) and BLM(-/-)LIG4(-/-) cells from the human pre-B cell line Nalm-6. BLM(-/-) cells exhibited retarded growth, increased mutation rates, and hypersensitivity to agents that block replication fork progression. Interestingly, these phenotypes were significantly suppressed by deletion of LIG4, suggesting that nonhomologous end-joining (NHEJ) is unfavorable for integrity and survival of cells lacking BLM. We propose that the absence of BLM leads to accumulation of replication-associated, one-ended DSBs, which are deleterious to cells and lead to genomic instability when repaired by NHEJ. In addition, the NHEJ pathway per se was marginally affected by BLM deficiency, as evidenced by x-ray sensitivity and I-SceI-based DSB repair assays. More intriguingly, however, these experiments revealed the presence of an alternative, DNA ligase IV-independent end-joining pathway, which was significantly affected by the loss of BLM. Collectively, our results provide the first evidence for genetic interactions between BLM and NHEJ in human cells.

Fanconi anemia (FA) is a human autosomal disorder characterized by cancer susceptibility and cellular sensitivity to DNA crosslinking agents such as mitomycin C and diepoxybutane. Six FA genes have been cloned including a gene designated XRCC9 (for X-ray Repair Cross Complementing), isolated using a mitomycin C-hypersensitive Chinese hamster cell mutant termed UV40, and subsequently found to be identical to FANCG. A nuclear complex containing the FANCA, FANCC, FANCE, FANCF and FANCG proteins is needed for the activation of a sixth FA protein FANCD2. When monoubiquitinated, the FANCD2 protein co-localizes with the breast cancer susceptibility protein BRCA1 in DNA damage induced foci. In this study, we have assigned NM3, a nitrogen mustard-hypersensitive Chinese hamster mutant to the same genetic complementation group as UV40. NM3, like human FA cell lines (but unlike UV40) exhibits a normal spontaneous level of sister chromatid exchange. We show that both NM3 and UV40 are also hypersensitive to other DNA crosslinking agents (including diepoxybutane and chlorambucil) and to non-crosslinking DNA damaging agents (including bleomycin, streptonigrin and EMS), and that all these sensitivities are all corrected upon transfection of the human FANCG/XRCC9 cDNA. Using immunoblotting, NM3 and UV40 were found not to express the active monoubiquitinated isoform of the FANCD2 protein, although expression of the FANCD-L isoform was restored in the FANCG cDNA transformants, correlating with the correction of mutagen-sensitivity. These data indicate that cellular resistance to these DNA damaging agents requires FANCG and that the FA gene pathway, via its activation of FANCD2 and that protein's subsequent interaction with BRCA1, is involved in maintaining genomic stability in response not only to DNA interstrand crosslinks but also a range of other DNA damages including DNA strand breaks. NM3 and other "FA-like" Chinese hamster mutants should provide an important resource for the study of these processes in mammalian cells.

PLK1 Acts in Homologous Recombinatorial Repair and in Mitosis As Synthetically Lethal with the Fanconi Anemia/BRCA Pathway

Sister chromatid exchange (SCE) can occur by several recombination mechanisms, including those directly initiated by double-strand breaks (DSBs), such as gap repair and break-induced replication (BIR), and those initiated when DNA polymerases stall, such as template switching. To elucidate SCE recombination mechanisms, we determined whether spontaneous and DNA damage-associated SCE requires specific genes within the RAD52 and RAD3 epistasis groups in Saccharomyces cerevisiae strains containing two his3 fragments, his3-Delta5' and his3-Delta3'::HOcs. SCE frequencies were measured after cells were exposed to UV, X-rays, 4-nitroquinoline 1-oxide (4-NQO) and methyl methanesulfonate (MMS), or when an HO endonuclease-induced DSB was introduced at his3-Delta3'::HOcs. Our data indicate that genes involved in gap repair, such as RAD55, RAD57 and RAD54, are required for DNA damage-associated SCE but not for spontaneous SCE. RAD50 and RAD59, genes required for BIR, are required for X-ray-associated SCE but not for SCE stimulated by HO-induced DSBs. In comparison with wild type, rates of spontaneous SCE are 10-fold lower in rad51 rad1 but not in either rad51 rad50 or rad51 rad59 double mutants. We propose that gap repair mechanisms are important in DNA damage-associated recombination, whereas alternative pathways, including a template switch pathway, play a role in spontaneous SCE.

DNA Interstrand crosslink (ICL) repair takes place at sites where the two strands of the DNA have become covalently linked by the byproducts of lipid peroxidation, endogenous aldehydes, and yet unknown metabolites. The repair of these lethal lesions takes place during DNA replication and requires a dual excision of the crosslinked bases and repair of the resulting double-strand breaks. This feat is accomplished in a multi-step process mediated by the Fanconi anemia (FA) pathway and factors that promote Homologous Recombination (HR), including BRCA1 and BRCA2 (reviewed in (1)). FA patients lack components of this pathway and suffer from bone marrow failure and infertility presumably due to the inability to maintain hematopoietic and germline stem cells. FA is also associated with a very high incidence of cancer, most likely due to the mutagenic nature of incompletely repaired ICLs. On the other hand, the induction of ICL is a major method of cancer treatment and often leads to excellent outcomes (e.g. cisplatin treatment of testicular cancer). Major effort has been directed into the identification of all of the components of the ICL repair pathways and the elucidation of the mechanism of such repair in the hopes of understanding how it suppresses tumorigenesis. Fanconi anemia registry (IFAR) housed at the Rockefeller University has been collecting patient samples and information since 1982. Using candidate gene sequencing of patient samples from the IFAR, we first found SLX4 as a new FA gene. SLX4 is a scaffold protein that interacts with three nucleases: XPF, MUS81, and SLX1 (2-4). We showed that this “Swiss army knife of repair” is mutated in patients with typical features of Fanconi anemia (5). Using SLX4 null patient cell lines, we explored the nuclease requirements in different DNA repair pathways during DNA replication and showed that the SLX4-bound XPF nuclease is essential for the ICL repair (6). This has been recently validated by work that identified XPF as a gene mutated in FA (7). In a separate study, we demonstrated that SLX4 is an important regulator of processing of replication intermediates including the Holliday junctions even under conditions of unperturbed DNA replication (8). This activity, although important for normal cellular growth, can be very mutagenic and thus cancer inducing. In our newest work, we have concentrated on a 12-year-old patient who was born with radial dysplasia, absent right thumb and tethered cord; the patient also exhibited enuresis and several absent permanent teeth. Marrow function is normal to date. No mutations in any of the known FA genes were identified. Whole exome sequencing identified an unexpected novel de novo mutation in RAD51. RAD51 is the mammalian homolog of RecA, mediating the homology search during HR. Although RAD51 foci formation is reduced and delayed following treatment with a range of DNA damaging agents, the patient fibroblasts do not exhibit sensitivity to ionizing radiation. In addition, the patient fibroblasts show wild type levels of sister chromatid exchanges following mitomycin C treatment, and are capable of performing HR for repairing dysfunctional GFP gene, as assessed by the DR-GFP assay. Collectively, these results suggest HR repair of double strand breaks is not impaired in the patient cells. Patient fibroblasts are hypersensitive to crosslinking agents and PARP inhibition. Following mitomycin C treatment, the cells exhibit elevated levels of both RPA foci formation and phosphorylation, indicating that RAD51 inhibits resection of nascent DNA strands, thereby stabilizing the ICL for its subsequent repair. Our study of the RAD51 patient mutation identifies the essential function of RAD51 at sites of stalled replication forks that is critical for the accurate repair of ICLs. Our results also suggest that the tumor suppressive role of RAD51-interacting proteins, including BRCA2, RAD51C, and RAD51D (all implicated in breast and ovarian cancer development) may exert their effect through this stabilizing activity of RAD51 at stalled replication forks.

Recent studies show overlap between Fanconi anemia (FA) proteins and those involved in DNA repair mediated by homologous recombination (HR). However, the mechanism by which FA proteins affect HR is unclear. FA proteins (FancA/C/E/F/G/L) form a multiprotein complex, which is responsible for DNA damage-induced FancD2 monoubiquitination, a key event for cellular resistance to DNA damage. Here, we show that FANCD2-disrupted DT40 chicken B-cell line is defective in HR-mediated DNA double-strand break (DSB) repair, as well as gene conversion at the immunoglobulin light-chain locus, an event also mediated by HR. Gene conversions occurring in mutant cells were associated with decreased nontemplated mutations. In contrast to these defects, we also found increased spontaneous sister chromatid exchange (SCE) and intact Rad51 foci formation after DNA damage. Thus, we propose that FancD2 promotes a subpathway of HR that normally mediates gene conversion by a mechanism that avoids crossing over and hence SCEs.

Fanconi anemia (FA) is a heterogeneous recessive disorder associated with a markedly elevated risk to develop cancer. To date sixteen FA genes have been identified, three of which predispose heterozygous mutation carriers to breast cancer. The FA proteins work together in a genome maintenance pathway, the so-called FA/BRCA pathway which is important during the S phase of the cell cycle. Since not all FA patients can be linked to (one of) the sixteen known complementation groups, new FA genes remain to be identified. In addition the complex FA network remains to be further unravelled. One of the FA genes, FANCI, has been identified via a combination of bioinformatic techniques exploiting FA protein properties and genetic linkage. The aim of this study was to develop a prioritization approach for proteins of the entire human proteome that potentially interact with the FA/BRCA pathway or are novel candidate FA genes. To this end, we combined the original bioinformatics approach based on the properties of the first thirteen FA proteins identified with publicly available tools for protein-protein interactions, literature mining (Nermal) and a protein function prediction tool (FuncNet). Importantly, the three newest FA proteins FANCO/RAD51C, FANCP/SLX4, and XRCC2 displayed scores in the range of the already known FA proteins. Likewise, a prime candidate FA gene based on next generation sequencing and having a very low score was subsequently disproven by functional studies for the FA phenotype. Furthermore, the approach strongly enriches for GO terms such as DNA repair, response to DNA damage stimulus, and cell cycle-regulated genes. Additionally, overlaying the top 150 with a haploinsufficiency probability score, renders the approach more tailored for identifying breast cancer related genes. This approach may be useful for prioritization of putative novel FA or breast cancer genes from next generation sequencing efforts.

Sister chromatid exchange in normal and Ph 1-positive leukemic cells after mitomycin-C treatment in vitro

BackgroundThe presence of inverted repeats (IRs) in DNA poses an obstacle to the normal progression of the DNA replication machinery, because these sequences can form secondary structures ahead of the replication fork. A failure to process and to restart the stalled replication machinery can lead to the loss of genome integrity. Consistently, IRs have been found to be associated with a high level of genome rearrangements, including deletions, translocations, inversions, and a high rate of sister-chromatid exchange (SCE). The RecQ helicase Sgs1, in Saccharomyces cerevisiae, is believed to act on stalled replication forks. To determine the role of Sgs1 when the replication machinery stalls at the secondary structure, we measured the rates of IR-associated and non-IR-associated spontaneous unequal SCE events in the sgs1 mutant, and in strains bearing mutations in genes that are functionally related to SGS1.ResultsThe rate of SCE in sgs1 cells for both IR and non-IR-containing substrates was higher than the rate in the wild-type background. The srs2 and mus81 mutations had modest effects, compared to sgs1. The exo1 mutation increased SCE rates for both substrates. The sgs1 exo1 double mutant exhibited synergistic effects on spontaneous SCE. The IR-associated SCE events in sgs1 cells were partially MSH2-dependent.ConclusionsThese results suggest that Sgs1 suppresses spontaneous unequal SCE, and SGS1 and EXO1 regulate spontaneous SCE by independent mechanisms. The mismatch repair proteins, in contradistinction to their roles in mutation avoidance, promote secondary structure-associated genetic instability.

Abstract. Sister chromatid exchange (SCE) is one of the cytogenetic methods which diagnoses damage to chromosomes and allows evaluation of the mutagenic influence of a given factor on a cell’s DNA. The purpose of the experiment was to determine the level of spontaneous and inductive SCE in the domestic cat. The research was carried out on 23 domestic cats Felis catus. Chromosome preparations were prepared from lymphocytes of peripheral blood after 72 h of in vitro breeding with the addition of bromodeoxyuridine (BrdU) in five different concentrations: 0.25, 0.5, 1.0, 2.5, 5.0 μg/ml. Chromosomes were stained by means of the fluorescence plus Giemsa (FPG) technique in order to carry out microscopic analysis. It was stated that the level of spontaneous SCE in the domestic cat occurs at a concentration 0.5 μg/ml on the basis of research previously carried out. Higher concentrations of this substance have a genotoxic action and damage DNA of chromosomes and induct additional SCEs in chromosomes of this species. Moreover, it was stated that the number of SCEs is higher in males than females. Our research also proved that the number of exchanges increases along with age in cats of both sexes.

Eight of the Fanconi anemia (FA) proteins form a core complex that activates the FA pathway. Some core complex components also form subcomplexes for yet-to-be-elucidated functions. Here, we have analyzed the interaction between a cytoplasmic FA subcomplex and the leukemic nucleophosmin (NPMc). Exogenous NPMc was degraded rapidly in FA acute myeloid leukemia bone marrow cells. Knockdown of FANCA or FANCC in leukemic OCI/AML3 cells induced rapid degradation of endogenous NPMc. NPMc degradation was mediated by the ubiquitin-proteasome pathway involving the IBR-type RING-finger E3 ubiquitin ligase IBRDC2, and genetic correction of FA-A or FA-C lymphoblasts prevented NPMc ubiquitination. Moreover, cytoplasmic FANCA and FANCC formed a cytoplasmic complex and interacted with NPMc. Using a patient-derived FANCC mutant and a nuclearized FANCC, we demonstrated that the cytoplasmic FANCA-FANCC complex was essential for NPMc stability. Finally, depletion of FANCA and FANCC in NPMc-positive leukemic cells significantly increased inflammation and chemoresistance through NF-κB activation. Our findings not only reveal the molecular mechanism involving cytoplasmic retention of NPMc but also suggest cytoplasmic function of FANCA and FANCC in NPMc-related leukemogenesis.

Objectives BLM , a member of the RecQ helicase family, plays an important role in DNA repair, and its biallelic mutations cause autosomal recessive Bloom syndrome, a disease characterized by elevated levels of sister chromatid exchange (SCE) in affected individuals and hereditary cancer susceptibility in carriers. This study aims to investigate genomic instability in breast cancer patients carrying heterozygous variants in the BLM gene. Methods Spontaneous chromosome breakage count and SCE counting were performed on newly drawn blood cultures, both spontaneous and stimulated. The spontaneous breakage count was conducted alongside control samples. In SCE analysis, 0–10 per metaphase was considered normal, 10–40 borderline, and counts above 40 were considered high. Results The study included 26 patients and one healthy control at each session. The clinical and pathological characteristics of the patients were evaluated. The analyses revealed borderline-level increased SCE rates in only one patient. No increase in spontaneous breakage count or SCE analysis was observed in other individuals compared to controls. Conclusions Increased genomic instability was not observed in the analyzed patient group. These results can lead to multiple interpretations. The variants carried in the BLM gene in the patient group may be of low pathogenicity, or increased instability compared to controls may not be necessary for heterozygous variants. Additionally, our patient group may not have been exposed to a genotoxic effect causing genomic instability. These results could also indicate a favorable position in terms of avoiding chemotherapy and radiotherapy complications.