Monoubiquitination of the Fanconi anaemia protein FANCD2 is a key event leading to repair of interstrand cross-links. It was reported earlier that FANCD2 co-localizes with NBS1. However, the functional connection between FANCD2 and MRE11 is poorly understood. In this study, we show that inhibition of MRE11, NBS1 or RAD50 leads to a destabilization of FANCD2. FANCD2 accumulated from mid-S to G2 phase within sites containing single-stranded DNA (ssDNA) intermediates, or at sites of DNA damage, such as those created by restriction endonucleases and laser irradiation. Purified FANCD2, a ring-like particle by electron microscopy, preferentially bound ssDNA over various DNA substrates. Inhibition of MRE11 nuclease activity by Mirin decreased the number of FANCD2 foci formed in vivo. We propose that FANCD2 binds to ssDNA arising from MRE11-processed DNA double-strand breaks. Our data establish MRN as a crucial regulator of FANCD2 stability and function in the DNA damage response.

The model carcinogen 4-nitroquinoline 1-oxide (4-NQO) has historically been characterized as "UV-mimetic" with respect to its genotoxic properties. However, recent evidence indicates that 4-NQO, unlike 254-nm UV light, may exert significant cytotoxic and/or mutagenic potential via the generation of reactive oxygen species. To elucidate the response of eukaryotic cells to 4-NQOinduced oxidative stress, we isolated Saccharomyces cerevisiae mutants exhibiting hypersensitivity to the cytotoxic effects of this mutagen. One such mutant, EBY1, was cross-sensitive to the oxidative agents UVA and diamide while retaining parental sensitivities to 254-nm UV light, methyl methanesulfonate, and ionizing radiation. A complementing gene (designated yPTPA1), restoring full UVA and 4-NQO resistance to EBY1 and encoding a protein that shares 40% identity with the human phosphotyrosyl phosphatase activator hPTPA, has been isolated. Targeted deletion of yPTPA1 in wild type yeast engendered the identical pattern of mutagen hypersensitivity as that manifested by EBY1, in addition to a spontaneous mutator phenotype that was markedly enhanced upon exposure to either UVA or 4-NQO but not to 254-nm UV or methyl methanesulfonate. Moreover, the yptpa1 deletion mutant exhibited a marked deficiency in the recovery of high molecular weight DNA following 4-NQO exposure, revealing a defect at the level of DNA repair. These data (i) strongly support a role for active oxygen intermediates in determining the genotoxic outcome of 4-NQO exposure and (ii) suggest a novel mechanism in yeast involving yPtpa1p-mediated activation of a phosphatase that participates in the repair of oxidative DNA damage, implying that hPTPA may exert a similar function in humans.Treatment with the agent 4-nitroquinoline 1-oxide (4-NQO) 1 has been widely employed in mammalian systems as a paradigm for DNA damage-induced carcinogenesis. To exert its neoplastic effect, 4-NQO must first undergo metabolic activation to the proximate carcinogen 4-hydroxyaminoquinoline 1-oxide, which, following acylation, reacts with DNA to form stable quinoline-purine monoadducts, i.e. at the exocyclic N-2 and N-6 positions of guanine and adenine, respectively (1, 2). In bacteria, yeast, and mammalian cells, these genotoxic "bulky" DNA lesions are processed largely by the nucleotide excision repair (NER) pathway in a manner analogous to classical dipyrimidine photoproducts (viz. cyclobutane pyrimidine dimers and (6 -4) pyrimidine-pyrimidone photoproducts) generated by the model DNA-damaging agent 254-nm UV light (3, 4). As such, mutants that are deficient in NER are hypersensitive to the genotoxic effects of 254-nm UV light, as well as 4-NQO (3-6). The apparent strong similarity in modes of cellular processing for 254-nm UV light-and 4-NQO-induced DNA damage in diverse prokaryotic and eukaryotic systems has often resulted in categorization of the latter agent as "UV-mimetic" (7).However, this designation may be inappropriate, because several recent investigations have clearly demonstrated that ...

The function of the Fanconi anemia group C protein (FANCC) is still unknown, though many studies point to a role in damage response signaling. Unlike other known FA proteins, FANCC is mainly localized to the cytoplasm and is thought to act as a messenger of cellular damage rather than an effector of repair. FANCC has been shown to interact with several cytoplasmic and nuclear proteins and to delay the onset of apoptosis through redox regulation of GSTP1. We investigated the fate and function of FANCC during apoptosis. Here we show that FANCC undergoes proteolytic modification by a caspase into a predominant 47-kDa ubiquitinated protein fragment. Lack of proteolytic modification at the putative cleavage site delays apoptosis but does not affect MMC complementation. These results suggest that FANCC function is regulated through proteolytic processing.Fanconi anemia (FA) 1 is a genetic disease identified as a bone marrow failure syndrome associated with cancer susceptibility, congenital defects, and cellular sensitivity to DNA cross-linking agents (1). FA patients are distributed into at least nine complementation groups. Eight FA genes have been cloned, FANCA, FANCC, FANCD2, FANCE, FANCF, FANCG, and FANCL, and recently the breast cancer susceptibility gene, BRCA2, has been assigned to complementation group D1 and possibly to group B, although the latter requires confirmation (2-11). Since a defect in any of the nine possible FA genes leads to a similar clinical phenotype, FA proteins appear to act in a common pathway to the breast cancer susceptibility proteins BRCA1 and BRCA2 sequentially and/or as a multiprotein complex.Several studies have shown that five of eight known FA proteins, FANCA, FANCC, FANCE, FANCF, and FANCG, bind to each other and form a multiprotein complex that translocates to the nucleus (12-18). Assembly of the multiprotein complex seems to be necessary for the activation through posttranslational monoubiquitination of FANCD2 (19,20). Monoubiquitination of FANCD2 is dependent on the recently identified FANCL gene that possesses ubiquitin ligase activity (11). The FANCD2 protein has been associated in nuclear foci with BRCA1, suggesting that BRCA1 may be a downstream component of the FA molecular pathway. Recently, the BRCA1 protein was shown to interact with the FANCA protein supporting a role of BRCA1 in the Fanconi pathway (21).The FA group D1 protein, BRCA2/FANCD1/FANCB, may function upstream in the pathway by promoting the formation of the FA complex and/or downstream by transducing signals from FA proteins to the DNA repair machinery. Mutation in one of the FA genes abolishes the FA complex formation, the monoubiquitination of FANCD2 and the nuclear foci formation in response to genotoxic stress. Nuclear localization of the FA complex was found to be critical for cellular resistance to MMC (17,22). However, several lines of investigation have suggested additional extranuclear functions for at least some FA components. For instance, FANCC and FANCG were shown to bind non-nuclear protein...