Deficiency in either of the breast cancer susceptibility proteins BRCA1 or BRCA2 induces profound cellular sensitivity to the inhibition of poly(ADP-ribose) polymerase (PARP) activity. We hypothesized that the critical role of BRCA1 and BRCA2 in the repair of double-strand breaks by homologous recombination (HR) was the underlying reason for this sensitivity. Here, we examine the effects of deficiency of several proteins involved in HR on sensitivity to PARP inhibition. We show that deficiency of RAD51, RAD54, DSS1, RPA1, NBS1, ATR, ATM, CHK1, CHK2, FANCD2, FANCA, or FANCC induces such sensitivity. This suggests that BRCA-deficient cells are, at least in part, sensitive to PARP inhibition because of HR deficiency. These results indicate that PARP inhibition might be a useful therapeutic strategy not only for the treatment of BRCA mutation-associated tumors but also for the treatment of a wider range of tumors bearing a variety of deficiencies in the HR pathway or displaying properties of 'BRCAness. ' (Cancer Res 2006; 66(16): 8109-15)

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.

Ataxia-telangiectasia (A-T) and Nijmegen breakage syndrome (NBS) are recessive genetic disorders with susceptibility to cancer and similar cellular phenotypes. The protein product of the gene responsible for A-T, designated ATM, is a member of a family of kinases characterized by a carboxy-terminal phosphatidylinositol 3-kinase-like domain. The NBS1 protein is specifically mutated in patients with Nijmegen breakage syndrome and forms a complex with the DNA repair proteins Rad50 and Mrel1. Here we show that phosphorylation of NBS1, induced by ionizing radiation, requires catalytically active ATM. Complexes containing ATM and NBS1 exist in vivo in both untreated cells and cells treated with ionizing radiation. We have identified two residues of NBS1, Ser 278 and Ser 343 that are phosphorylated in vitro by ATM and whose modification in vivo is essential for the cellular response to DNA damage. This response includes S-phase checkpoint activation, formation of the NBS1/Mrel1/Rad50 nuclear foci and rescue of hypersensitivity to ionizing radiation. Together, these results demonstrate a biochemical link between cell-cycle checkpoints activated by DNA damage and DNA repair in two genetic diseases with overlapping phenotypes.

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.

We show here that the radiosensitive Chinese hamster cell mutant (V-C8) of group XRCC11 is defective in the breast cancer susceptibility gene Brca2. The very complex phenotype of V-C8 cells is complemented by a single human chromosome 13 providing the BRCA2 gene, as well as by the murine Brca2 gene. The Brca2 deficiency in V-C8 cells causes hypersensitivity to various DNA-damaging agents with an extreme sensitivity toward interstrand DNA cross-linking agents. Furthermore, V-C8 cells show radioresistant DNA synthesis after ionizing radiation, suggesting that Brca2 deficiency affects cell cycle checkpoint regulation. In addition, V-C8 cells display tremendous chromosomal instability and a high frequency of abnormal centrosomes. The mutation spectrum at the hprt locus showed that the majority of spontaneous mutations in V-C8 cells are deletions, in contrast to wild-type V79 cells. A mechanistic explanation for the genome instability phenotype of Brca2-deficient cells is provided by the observation that the nuclear localization of the central DNA repair protein in homologous recombination, Rad51, is reduced in V-C8 cells.V-C8 is a Chinese hamster cell mutant that represents the XRCC11 complementation group, among X-ray-sensitive rodent cell mutants, as well as a distinguished group among mitomycin C (MMC)-sensitive rodent cell mutants (for a review, see reference 46). This mutant is extremely sensitive to cross-linking agents, but it also shows an increased sensitivity toward many other DNA-damaging agents, such as methyl methanesulfonate (MMS) and UV light (19,48). This suggests that the XRCC11 gene is involved in a wide-ranging cellular response induced by various types of DNA damage. V-C8 cells display radioresistant DNA synthesis (RDS) after ionizing irradiation (36), which is indicative of a defect in DNA damage recognition or cell cycle checkpoint regulation. However, the high level of spontaneous and cross-link-induced chromosomal aberrations manifested by V-C8 cells (19) indicates a possible defect in DNA repair. Indeed, V-C8 cells have an impaired capacity for repair of DNA double-strand breaks (DSBs) after irradiation (36).In mammalian cells, DSBs are repaired via either nonhomologous end joining (NHEJ) or homologous recombination (HR) (reviewed in reference 14). Genetic complementation studies have determined that V-C8 cells are not defective in DNA-PKcs, Ku80, or Xrcc4, key components of NHEJ, nor in Xrcc2 or Xrcc3, proteins involved in HR, and that this mutant represents a separate complementation group, XRCC11 (36,46). When compared to cell lines defective in the above-mentioned proteins, the overall phenotype of V-C8 cells more closely resembles those of the Xrcc2-and Xrcc3-defective hamster cell lines irs1 and irs1SF, respectively (8,12,16). In common with V-C8, irs1 and irs1SF exhibit an extreme sensitivity to cross-linking agents that is not observed in cell lines defective in NHEJ proteins. This favors the hypothesis that the XRCC11 gene defective in V-C8 cells might be involved in HR. A key playe...

Radiosensitive T -B -severe combined immunodeficiency (RS-SCID) is caused by defects in the nonhomologous end-joining (NHEJ) DNA repair pathway, which results in failure of functional V(D)J recombination. Here we have identified the first human RS-SCID patient to our knowledge with a DNA-PKcs missense mutation (L3062R). The causative mutation did not affect the kinase activity or DNA end-binding capacity of DNA-PKcs itself; rather, the presence of long P-nucleotide stretches in the immunoglobulin coding joints indicated that it caused insufficient Artemis activation, something that is dependent on Artemis interaction with autophosphorylated DNA-PKcs. Moreover, overall end-joining activity was hampered, suggesting that Artemis-independent DNA-PKcs functions were also inhibited. This study demonstrates that the presence of DNA-PKcs kinase activity is not sufficient to rule out a defect in this gene during diagnosis and treatment of RS-SCID patients. Further, the data suggest that residual DNA-PKcs activity is indispensable in humans.Introduction SCID is an inherited primary immunodeficiency. SCID patients present in the first year of life with severe opportunistic infections, chronic diarrhea, and failure to thrive. The total group of SCID patients can be divided in 2 main categories: those with T -B + SCID, who have a T cell signaling defect (70%), and those with T -B -SCID, who have a defect in V(D)J recombination (30%). V(D)J recombination assembles variable (V), diversity (D), and joining (J) gene segments of the Ig and TCR genes during B and T cell differentiation in order to generate a broad repertoire of antigen-specific receptors. V(D)J recombination starts with introduction of DNA breaks at the border of the gene segments and the flanking recombination signal sequences (RSSs) by the RAG1 and RAG2 proteins (1). The resulting blunt signal ends are ligated directly, forming a signal joint. The hairpin sealed coding ends require further processing before coding joint formation can occur. Recognition and repair of the DNA ends occur via the general nonhomologous end-joining (NHEJ) pathway of DNA double-strand break (DSB) repair (2, 3).DSBs induce ATM kinase activity, which phosphorylates histone H2AX (4), followed by binding of 53BP1, MDC1, and a complex of MRE11, RAD50, and NBS1 (MRN complex) (5, 6). These proteins form a microenvironment that holds together the DNA ends over a relatively large distance but still allows some degree of freedom for movement of the DNA ends and access of NHEJ proteins (7).

Artesunate is a semisynthetic derivative from artemisinin, a natural product from the Chinese herb Artemisia annua L. It exerts antimalarial activity, and, additionally, artemisinin and its derivatives are active against cancer cells. The active moiety is an endoperoxide bridge. Its cleavage leads to the formation of reactive oxygen species and carbon-centered radicals. These highly reactive molecules target several proteins in Plasmodia, which is thought to result in killing of the microorganism. DNA damage induced by artemisinins has not yet been described. Here, we show that artesunate induces apoptosis and necrosis. It also induces DNA breakage in a dose-dependent manner as shown by single-cell gel electrophoresis. This genotoxic effect was confirmed by measuring the level of ;-H2AX, which is considered to be an indication of DNA double-strand breaks (DSB). Polymerase B-deficient cells were more sensitive than the wild-type to artesunate, indicating that the drug induces DNA damage that is repaired by base excision repair. irs1 and VC8 cells defective in homologous recombination (HR) due to inactivation of XRCC2 and BRCA2, respectively, were more sensitive to artesunate than the corresponding wild-type. This was also true for XR-V15B cells defective in nonhomologous endjoining (NHEJ) due to inactivation of Ku80. The data indicate that DSBs induced by artesunate are repaired by the HR and NHEJ pathways. They suggest that DNA damage induced by artesunate contributes to its therapeutic effect against cancer cells. [Cancer Res 2008;68(11):4347-51]

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