Rad51C is essential for embryonic development and haploinsufficiency causes increased DNA damage sensitivity and genomic instability.

Homologous recombination is essential for repair of DNA interstrand cross-links and double-strand breaks. The Rad51C protein is one of the five Rad51 paralogs in vertebrates implicated in homologous recombination. A previously described hamster cell mutant defective in Rad51C (CL-V4B) showed increased sensitivity to DNA damaging agents and displayed genomic instability. Here, we identified a splice donor mutation at position +5 of intron 5 of the Rad51C gene in this mutant, and generated mice harboring an analogous base pair alteration. Rad51C(splice) heterozygous animals are viable and do not display any phenotypic abnormalities, however homozygous Rad51C(splice) embryos die during early development (E8.5). Detailed analysis of two CL-V4B revertants, V4B-MR1 and V4B-MR2, that have reduced levels of full-length Rad51C transcript when compared to wild type hamster cells, showed increased sensitivity to mitomycin C (MMC) in clonogenic survival, suggesting haploinsufficiency of Rad51C. Similarly, mouse Rad51C(splice/neo) heterozygous ES cells also displayed increased MMC sensitivity. Moreover, in both hamster revertants, Rad51C haploinsufficiency gives rise to increased frequencies of spontaneous and MMC-induced chromosomal aberrations, impaired sister chromatid cohesion and reduced cloning efficiency. These results imply that adequate expression of Rad51C in mammalian cells is essential for maintaining genomic stability and sister chromatid cohesion to prevent malignant transformation.

Warsaw breakage syndrome, a cohesinopathy associated with mutations in the XPD helicase family member DDX11/ChlR1.

The iron-sulfur-containing DNA helicases XPD, FANCJ, DDX11, and RTEL represent a small subclass of superfamily 2 helicases. XPD and FANCJ have been connected to the genetic instability syndromes xeroderma pigmentosum and Fanconi anemia. Here, we report a human individual with biallelic mutations in DDX11. Defective DDX11 is associated with a unique cellular phenotype in which features of Fanconi anemia (drug-induced chromosomal breakage) and Roberts syndrome (sister chromatid cohesion defects) coexist. The DDX11-deficient patient represents another cohesinopathy, besides Cornelia de Lange syndrome and Roberts syndrome, and shows that DDX11 functions at the interface between DNA repair and sister chromatid cohesion.

The cellular phenotype of Roberts syndrome fibroblasts as revealed by ectopic expression of ESCO2.

Cohesion between sister chromatids is essential for faithful chromosome segregation. In budding yeast, the acetyltransferase Eco1/Ctf7 establishes cohesion during DNA replication in S phase and in response to DNA double strand breaks in G2/M phase. In humans two Eco1 orthologs exist: ESCO1 and ESCO2. Both proteins are required for proper sister chromatid cohesion, but their exact function is unclear at present. Since ESCO2 has been identified as the gene defective in the rare autosomal recessive cohesinopathy Roberts syndrome (RBS), cells from RBS patients can be used to elucidate the role of ESCO2. We investigated for the first time RBS cells in comparison to isogenic controls that stably express V5- or GFP-tagged ESCO2. We show that the sister chromatid cohesion defect in the transfected cell lines is rescued and suggest that ESCO2 is regulated by proteasomal degradation in a cell cycle-dependent manner. In comparison to the corrected cells RBS cells were hypersensitive to the DNA-damaging agents mitomycin C, camptothecin and etoposide, while no particular sensitivity to UV, ionizing radiation, hydroxyurea or aphidicolin was found. The cohesion defect of RBS cells and their hypersensitivity to DNA-damaging agents were not corrected by a patient-derived ESCO2 acetyltransferase mutant (W539G), indicating that the acetyltransferase activity of ESCO2 is essential for its function. In contrast to a previous study on cells from patients with Cornelia de Lange syndrome, another cohesinopathy, RBS cells failed to exhibit excessive chromosome aberrations after irradiation in G2 phase of the cell cycle. Our results point at an S phase-specific role for ESCO2 in the maintenance of genome stability.

Impaired FANCD2 monoubiquitination and hypersensitivity to camptothecin uniquely characterize Fanconi anemia complementation group M.

FANCM is a component of the Fanconi anemia (FA) core complex and one FA patient (EUFA867) with biallelic mutations in FANCM has been described. Strikingly, we found that EUFA867 also carries biallelic mutations in FANCA. After correcting the FANCA defect in EUFA867 lymphoblasts, a "clean" FA-M cell line was generated. These cells were hypersensitive to mitomycin C, but unlike cells defective in other core complex members, FANCM(-/-) cells were proficient in monoubiquitinating FANCD2 and were sensitive to the topoisomerase inhibitor camptothecin, a feature shared only with the FA subtype D1 and N. In addition, FANCM(-/-) cells were sensitive to UV light. FANCM and a C-terminal deletion mutant rescued the cross-linker sensitivity of FANCM(-/-) cells, whereas a FANCM ATPase mutant did not. Because both mutants restored the formation of FANCD2 foci, we conclude that FANCM functions in an FA core complex-dependent and -independent manner.

Increased DNA damage sensitivity of Cornelia de Lange syndrome cells: evidence for impaired recombinational repair.

Cornelia de Lange syndrome (CdLS) is a rare dominantly inherited multisystem disorder affecting both physical and mental development. Heterozygous mutations in the NIPBL gene were found in about half of CdLS cases. Scc2, the fungal ortholog of the NIPBL gene product, is essential for establishing sister chromatid cohesion. In yeast, the absence of cohesion leads to chromosome mis-segregation and defective repair of DNA double-strand breaks. To evaluate possible DNA repair defects in CdLS cells, we characterized the cellular responses to DNA-damaging agents. We show that cells derived from CdLS patients, both with and without detectable NIPBL mutations, have an increased sensitivity for mitomycin C (MMC). Exposure of CdLS fibroblast and B-lymphoblastoid cells to MMC leads to enhanced cell killing and reduced proliferation and, in the case of primary fibroblasts, an increased number of chromosomal aberrations. After X-ray exposure increased numbers of chromosomal aberrations were also detected, but only in cells irradiated in the G(2)-phase of the cell cycle when repair of double-strand breaks is dependent on the establishment of sister chromatid cohesion. Repair at the G(1) stage is not affected in CdLS cells. Our studies indicate that CdLS cells have a reduced capacity to tolerate DNA damage, presumably as a result of reduced DNA repair through homologous recombination.

Cellular characterization of cells from the Fanconi anemia complementation group, FA-D1/BRCA2.

Fanconi anemia (FA) is an inherited cancer-susceptibility disorder, characterized by genomic instability and hypersensitivity to DNA cross-linking agents. The discovery of biallelic BRCA2 mutations in the FA-D1 complementation group allows for the first time to study the characteristics of primary BRCA2-deficient human cells. FANCD1/BRCA2-deficient fibroblasts appeared hypersensitive to mitomycin C (MMC), slightly sensitive to methyl methane sulfonate (MMS), and like cells derived from other FA complementation groups, not sensitive to X-ray irradiation. However, unlike other FA cells, FA-D1 cells were slightly sensitive to UV irradiation. Despite the observed lack of X-ray sensitivity in cell survival, significant radioresistant DNA synthesis (RDS) was observed in the BRCA2-deficient fibroblasts but also in the FANCA-deficient fibroblasts, suggesting an impaired S-phase checkpoint. FA-D1/BRCA2 cells displayed greatly enhanced levels of spontaneous as well as MMC-induced chromosomal aberrations (CA), similar to cells deficient in homologous recombination (HR) and non-D1 FA cells. In contrast to Brca2-deficient rodent cells, FA-D1/BRCA2 cells showed normal sister chromatid exchange (SCE) levels, both spontaneous as well as after MMC treatment. Hence, these data indicate that human cells with biallelic BRCA2 mutations display typical features of both FA- and HR-deficient cells, which suggests that FANCD1/BRCA2 is part of the integrated FA/BRCA DNA damage response pathway but also controls other functions outside the FA pathway.

Chinese hamster cell mutant, V-C8, a model for analysis of Brca2 function.

The previously described Chinese hamster cell mutant V-C8 that is defective in Brca2 shows a very complex phenotype, including increased sensitivity towards a wide variety of DNA damaging agents, chromosomal instability, abnormal centrosomes and impaired formation of Rad51 foci in response to DNA damage. Here, we demonstrate that V-C8 cells display biallelic nonsense mutations in Brca2, one in exon 15 and the other in exon 16, both resulting in truncated Brca2 proteins. We generated several independent mitomycin C (MMC)-resistant clones from V-C8 cells that had acquired an additional mutation leading to the restoration of the open reading frame of one of the Brca2 alleles. In two of these revertants, V-C8-Rev 1 and V-C8-Rev 6, the reversions lead to the wild-type Brca2 sequence. The V-C8 revertants did not gain the entire wild-type phenotype and still show a 2.5-fold increased sensitivity to mitomycin C (MMC), higher levels of spontaneous and MMC-induced chromosomal aberrations, as well as abnormal centrosomes when compared to wild-type cells. Our results suggest that Brca2 heterozygosity in hamster cells primarily gives rise to sensitivity to DNA cross-linking agents, especially chromosomal instability, a feature that might also be displayed in BRCA2 heterozygous mutation carriers.

Inducibility of nuclear Rad51 foci after DNA damage distinguishes all Fanconi anemia complementation groups from D1/BRCA2.

Fanconi anemia (FA) is a cancer susceptibility disorder characterized by chromosomal instability and hypersensitivity to DNA cross-linking agents. So far 11 complementation groups have been identified, from which only FA-D1/BRCA2 and FA-J are defective downstream of the central FANCD2 protein as cells from these groups are capable of monoubiquitinating FANCD2. In this study we show that cells derived from patients from the new complementation groups, FA-I, FA-J and FA-L are all proficient in DNA damage induced Rad51 foci formation, making the cells from FA-D1/BRCA2 patients that are defective in this process the sole exception. Although FA-B patient HSC230 was previously reported to also have biallelic BRCA2 mutations, we found normal Rad51 foci formation in cells from this patient, consistent with the recent identification of an X-linked gene being mutated in four unrelated FA-B patients. Thus, our data show that none of the FA proteins, except BRCA2, are required to sequester Rad51 into nuclear foci. Since cells from the FA-D1 and FA-J patient groups are both able to monoubiquitinate FANCD2, the "Rad51 foci phenotype" provides a convenient assay to distinguish between these two groups. Our results suggest that FANCJ and FANCD1/BRCA2 are part of the integrated FANC/BRCA DNA damage response pathway or, alternatively, that they represent sub-pathways in which only FANCD1/BRCA2 is directly connected to the process of homologous recombination.

The DNA helicase BRIP1 is defective in Fanconi anemia complementation group J.

The protein predicted to be defective in individuals with Fanconi anemia complementation group J (FA-J), FANCJ, is a missing component in the Fanconi anemia pathway of genome maintenance. Here we identify pathogenic mutations in eight individuals with FA-J in the gene encoding the DEAH-box DNA helicase BRIP1, also called FANCJ. This finding is compelling evidence that the Fanconi anemia pathway functions through a direct physical interaction with DNA.

Impaired DNA damage-induced nuclear Rad51 foci formation uniquely characterizes Fanconi anemia group D1.

Fanconi anemia is a hereditary cancer susceptibility disorder characterized at the cellular level by spontaneous chromosomal instability and specific hypersensitivity to DNA cross-linking agents such as mitomycin C. This phenotype suggests a possible role for the Fanconi anemia proteins in the repair of DNA lesions induced by these agents, but the molecular mechanism underlying the defect in this disorder has not yet been identified. Here, we show that amongst eight so far identified complementation groups of Fanconi anemia, only fibroblasts derived from group D1 are defective in the formation of nuclear Rad51 foci after X-ray irradiation or mitomycin C treatment. This indicates that the FANCD1 gene product is uniquely involved in the assembly and/or stabilization of the Rad51 complex. Since DNA damage-induced Rad51 nuclear foci are thought to reflect repair of DNA double-strand breaks by homologous recombination, our results suggest that FANCD1 is likely to be involved in homologous recombination-dependent repair.

Mammalian Rad51C contributes to DNA cross-link resistance, sister chromatid cohesion and genomic stability.

The eukaryotic Rad51 protein is a structural and functional homolog of Escherichia coli RecA with a role in DNA repair and genetic recombination. Five paralogs of Rad51 have been identified in vertebrates, Rad51B, Rad51C, Rad51D, Xrcc2 and Xrcc3, which are also implicated in recombination and genome stability. Here, we identify a mammalian cell mutant in Rad51C. We show that the Chinese hamster cell mutant, CL-V4B, has a defect in Rad51C. Sequencing of the hamster Rad51C cDNA revealed a 132 bp deletion corresponding to an alternatively spliced transcript with lack of exon 5. CL-V4B was hypersensitive to the interstrand cross-linking agents mitomycin C (MMC) and cisplatinum, the alkylating agent methyl methanesulfonate and the topoisomerase I inhibitor campthotecin and showed impaired Rad51 foci formation in response to DNA damage. The defect in Rad51C also resulted in an increase of spontaneous and MMC-induced chromosomal aberrations as well as a lack of induction of sister chromatid exchanges. However, centrosome formation was not affected. Intriguingly, a reduced level of sister chromatid cohesion was found in CL-V4B cells. These results reveal a role for Rad51C that is unique among the Rad51 paralogs.

Reduced fertility and hypersensitivity to mitomycin C characterize Fancg/Xrcc9 null mice.

Fanconi anemia (FA) is a heterogeneous autosomal recessive chromosomal instability syndrome associated with diverse developmental abnormalities, progressive bone marrow failure and a predisposition to cancer. Spontaneous chromosomal breakage and hypersensitivity to DNA cross-linking agents characterize the cellular FA phenotype. The gene affected in FA complementation group G patients was initially identified as XRCC9, for its ability to partially correct the cellular phenotype of the Chinese hamster ovary (CHO) cell mutant UV40. By targeted disruption we generated Fancg/Xrcc9 null mice. Fancg knock-out (KO) mice were born at expected Mendelian frequencies and showed normal viability. In mice, functional loss of Fancg did not result in developmental abnormalities or a pronounced incidence of malignancies. During a 1 year follow-up, blood cell parameters of Fancg KO mice remained within normal values, revealing no signs of anemia. Male and female mice deficient in Fancg showed hypogonadism and impaired fertility, consistent with the phenotype of FA patients. Mouse embryonic fibroblasts (MEFs) from the KO animals exhibited the FA characteristic cellular response in showing enhanced spontaneous chromosomal instability and a hyper-responsiveness to the clastogenic and antiproliferative effects of the cross-linking agent mitomycin C (MMC). The sensitivity to UV, X-rays and methyl methanesulfonate, reported for the CHO mutant cell line UV40, was not observed in Fancg(-/-) MEFs. Despite a lack of hematopoietic failure in the KO mice, clonogenic survival of bone marrow cells in vitro was strongly reduced in the presence of MMC. The characteristics of the Fancg(-/-) mice closely resemble those reported for Fancc and Fanca null mice, supporting a tight interdependence of the corresponding gene products in a common pathway.