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.

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.

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 Ϫ/Ϫ 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 Ϫ/Ϫ cells were sensitive to UV light. IntroductionFanconi anemia (FA) is a recessive genetic instability syndrome that has uncovered a cellular pathway involved in the protection against replication-blocking lesions. Inactivation of this pathway, as seen in FA patients, results in hypersensitivity to DNA crosslinking agents and cancer susceptibility. 1 Defects in 13 different genes have been found in FA patients, 2 and the proteins encoded by these genes cooperate in a pathway that can be subdivided in an upstream and downstream part based upon the monoubiquitination of FANCD2 and FANCI. 1 The upstream part of the pathway consists of a nuclear core complex formed by the FA proteins FANCA, -B, -C, -E, -F, -G, -L, and -M and 2 FA-associated proteins FAAP100 and FAAP24. This complex monoubiquitinates FANCD2 through the E3-ubiquitin ligase FANCL in conjunction with the ubiquitin-conjugating enzyme. 3,4 The FA core complex, UBE2T, and FANCD2 are independently recruited to the stalled replication fork. 5 For FANCD2, this relies on the ATR-mediated phosphorylation of its binding partner FANCI, 6 whereas the recruitment of the FA core complex seems to depend on FANCM. 7 Like FANCD2, FANCI is also monoubiquitinated by the FA core complex and these modified proteins colocalize with Rad51 and BRCA1 in nuclear foci. 8,9 The link between FA and BRCA proteins was further strengthened by the discovery of FA patients with mutations in BRCA2, 10 and in the BRCA1-and BRCA2-interacting proteins BRIP1 11,12 and PALB2. 13,14 FA patients with a defect in any of these genes have normal FANCD2 monoubiquitination and therefore these proteins are considered as downstream players in the FA pathway.Despite the identification of the various components of the FA core complex, its role in the maintenance of genome stability remains unclear, because of the absence of functional domains in most of the core complex members. A notable exception is FANCM, an ortholog of the archaeal DNA repair protein HEF, which contains 2 conserved domains: a DEAH helicase domain in the N-terminus and an endonuclease domain in the C-terminus. 15,16 The helicase domain is shared with yeast orthologs MPH1 (Saccharomyces cerevisiae) and FML1 (Schizosaccharomyces pombe), which play a regulatory role in homologous recombination repair by replication fork reversal and D-loop disruption. [17][18][19] HEF and MPH1 possess helicase activity, 20,21 whereas for F...

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.

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.